Treatment of hyperproliferative disease with superantigens in combination with another anticancer agent

ABSTRACT

The present invention relates to methods of treating mammals affected by, for example, a hyperproliferative disease such as cancer, by administering a tumor-targeted superantigen and a chemotherapeutic agent, whereby the administration of the tumor-targeted superantigen and chemotherapeutic agent reduce the antibody response and enhance the T cell response. The superantigen, wild-type or modified, is fused to a target-seeking moiety, such as an antibody or an antibody active fragment. The combined administration of a superantigen and a chemotherapeutic agent provides enhanced therapeutic effects in a treated animal.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/202,507, now U.S. Pat. No. 7,763,253, filed Aug. 12, 2005, whichclaims benefit of U.S. Provisional Application No. 60/601,548, filedAug. 13, 2004, and of Swedish Patent Application Number SE 0402025-1,filed Aug. 17, 2004, each of which are incorporated herein by referencein their entireties.

This application is also related to the following U.S. patents andpatent applications, each of which are incorporated herein by referencein their entirety: U.S. Pat. Nos. 5,858,363, 6,197,299, 6,514,498, U.S.patent application Ser. Nos. 08/765,695 (filed Jul. 25, 1997),10/283,838 (filed Oct. 20, 2002) (U.S. Application Publication No.20030092894), 09/463,470 (filed Jan. 21, 2000), and 09/900,766 (filedJul. 6, 2001) (U.S. Application Publication No. 20030039655).

TECHNICAL FIELD

The present invention relates to methods of treating mammals, forexample humans, by administering a superantigen and an anticancer agent.More particularly, the invention relates to the administration of asuperantigen and an anticancer agent for the treatment of ahyperproliferative disease, for example, cancer. Still moreparticularly, the present invention relates to the administration of ananticancer agent, such as a chemotherapy drug, in sequenced dosage witha bacterial superantigen, such as a tumor-targeted superantigen (“TTS”),in the treatment of cancer, whereby the administration results in asynergistic effect compared to the administration of each agent alone.Still further, the present invention relates to a method ofadministering a superantigen, such as tumor-targeted superantigen and ananticancer agent, such as a cytostatic chemotherapeutic, whereby theadministration reduces the production of antibodies to the superantigenin the treated host, compared with the administration of superantigenalone. Embodiments of the present invention also relates to kitscontaining a superantigen, such as a tumor-targeted superantigen, and ananticancer agent, such as a chemotherapeutic drug.

BACKGROUND OF THE INVENTION

Developing effective treatments of malignant tumors is still achallenge, despite encouraging progress during the last decades. Today,the most commonly used treatment for advanced metastasized cancerinvolves the use of cytostatic drugs, sometimes one or more incombination. Cytostatic drugs affect proliferating cells by interferingwith fundamental processes of cell multiplication, thereby inducing cellarrest or cell death. Targets include DNA, nucleotide metabolism,enzymes related to DNA integrity, and the cytoskeleton. A group ofcytostatic drugs proven to be effective in the treatment of cancercomprises alkylating agents, antimetabolites, inhibitors of mitosis,cytostatic antibiotics, platinum based compounds and topoisomeraseinhibitors.

Superantigens are bacterial, viral proteins, and now, human-engineeredmolecules, capable of activating T lymphocytes at picomolarconcentrations. They bind directly to the major histocompatibilitycomplex (MHC) without being processed. Superantigens bind unprocessedoutside the antigen-binding groove on MHC class II molecules, therebyavoiding most of the polymorphism in the conventional peptide-bindingsite. Superantigens bind to the Vβ chain of the T cell receptor (TCR),instead of binding to the hypervariable loops of the T cell receptor(TCR) and activate T cells. Therefore, when a superantigen isadministered to an animal, such as a human, a subset of T cells isnon-specificially activated by the superantigen, as opposed toadministration of a “regular” antigen, which would specificiallyactivate only a small sub-set of T cells. Examples of bacterialsuperantigens include, but are not limited to, Staphylococcalenterotoxin (SE), Streptococcus pyogenes exotoxin (SPE), Staphylococcusaureus toxic shock-syndrome toxin (TSST-1), Streptococcal mitogenicexotoxin (SME), Streptococcal superantigen (SSA), Staphylococcalenterotoxin A (SEA), and Staphylococcal enterotoxin E (SEE).

As discussed in more detail below, and in the U.S. patent applicationsand patents noted and incorporated herein by reference, superantigenscan be modified, for example, by modifying the DNA sequences encodingsuperantigens, such that, for example, they encode modifiedsuperantigens having improved therapeutic properties. For example,modified superantigens can have reduced binding to MHC class II antigenscompared to unmodified wild-type superantigens, resulting in reducedsystemic toxicity, and/or can have reduced seroreactivity and/ordecreased ability to induce an antibody response compared to thewild-type superantigens, resulting in reduced encounters with, andpotential difficulties with, host antibodies. An example of a modifiedsuperantigen is SEA/E (e.g., SEA/E-120), described in detail below,which binds to MHC class II antigens to activate T cells (e.g., likethat of wild-type SEA), and has reduced seroreactivity (e.g., lower thanwild-type SEE).

In some embodiments of the present invention, a targeting moiety, forexample an antibody or antibody fragment, may be conjugated to asuperantigen (wild-type or modified), providing a targeted superantigen.If the antibody, or antibody fragment recognizes a tumor-associatedantigen, the targeted superantigen may be called a tumor-targetedsuperantigen (“TTS”). Targeted superantigens retain the ability toactivate large number of T lymphocytes, and add the ability to directthe activated lymphocytes to cells bearing the target moiety. Forexample, TTS molecules activate large numbers of T cells and direct themto tissues containing the tumor-associate antigen that is recognized bythe targeting moiety. By doing so, specific target cells can be killed,leaving the rest of the body relatively unharmed. Such “magic bullet”therapy is quite desired in the art, as non-specific anticancer agents,such as radiation, and cytostatic and cytotoxic chemotherapeutic drugs,are nonspecific and kill large numbers of cells that are not associatedwith tumors to be treated. For example, studies with TTS have show thatinflammation by cytotoxic T lymphocytes (CTLs) into tumor tissueincreases rapidly in response to the first injection of a targetedsuperantigen (Dohlsten et al., 1995). This inflammation withinfiltration of CTLs into the tumor is one of the major effectors of theanti-tumor therapeutic of targeted superantigens.

Anticancer agents, such as cytostatic drugs and radiation, generallywork by affecting or preventing cell division. Because they arenonspecific, for example, all dividing cells in a treated animal, suchas a human, are affected. This typically results in extreme adverse sideeffects from chemotherapy treatment, such as gastrological disturbances,loss of hair, and damage to the immune system, that are notoriouslywell-known, both to those skilled in the art as well as to thepopulation as a whole.

Many aspects of the immune system is characterized by dividing cells.For example, in an immune response, the requisite immunocyte expansionphase is characterized by immune cell proliferation. This proliferationis fundamental and essential to a productive immune response. Sincecytostatic agents affect dividing cells, cytostatic agents are known tobe deleterious to the immune system. In fact, a compromised immunesystem is one of the more common and serious side effects of treatingcancer with chemotherapeutic drugs.

On the other hand, immune therapy relies on a functional immune system.(Chen 2003; Le Poole et al., 2003). Immune therapies such as tumorvaccines rely on a functional immune response in a treated patient. Forexample, for a productive immune response to a tumor vaccine, T and Blymphocytes must be activated, expand and differentiate into sufficientnumbers of effector cells (Le Poole et al., 2003; Chen 2003). This ofcourse requires cell division. Further, it is highly probable that suchan immune response also requires that the immunocyte proliferationrepeat a second time in order to reach a productive antitumor response(Tester and Mora 2001).

One skilled in the art would, therefore, not expect immune therapy to becompatible with anticancer agents such as cytostatic agents that affectcell division. Immune therapy is expected to require cell division inthe immune system, and cytostatic agents are known to affect or preventcell division.

Therefore, prior to the advent of the instant invention, it was notexpected to be possible to combine immunotherapy with anticancer agents,such as cytostatic or cytocidal drugs. Applicants understand that thepresent invention is the first time that effective use has been made ofimmunotherapy in the context of anticancer treatment, such as withcytostatic or cytotoxic drugs. As explained below, the inventors havediscovered that not only is immunotherapy with superantigens compatiblewith anticancer agents such as cytostatic drugs, but in fact,coordinating the dosing of these two agents results in synergisticanticancer results. Furthermore, this combination therapy affords thebenefit of reducing the production of antibodies in a treated person tothe superantigens.

BRIEF SUMMARY OF THE INVENTION

The instant invention relates to a method of treating a mammal, such asa human, with superantigens and an anticancer agent, such as achemotherapeutic agent. The instant invention relates to the discoverythat superantigen therapy and other anticancer therapies can be combinedto result in synergistic treatment effects, and reduced antibodyproduction in the treated host to superantigen therapy.

One embodiment of the present invention comprises a method of reducingan antibody response to a tumor-targeted superantigen in a mammal,comprising systemically administering to the mammal a tumor-targetedsuperantigen and administering a chemotherapeutic agent. Thetumor-targeted superantigen is administered by an administrationselected from the group consisting of administration of thetumor-targeted superantigen prior to the administration of thechemotherapeutic agent, administration of the tumor-targetedsuperantigen after the administration of the chemotherapeutic agent,administration of the tumor-targeted superantigen during theadministration of the chemotherapeutic agent, and administration of thetumor-targeted superantigen between administration of thechemotherapeutic agent.

In certain embodiments, the tumor-targeted superantigen is selected fromthe group consisting of staphylococcal enterotoxin A, modifiedstaphylococcal enterotoxin A, staphylococcal enterotoxin E, and modifiedstaphylococcal enterotoxin E. More specifically, the tumor-targetedsuperantigen comprises a targeting moiety selected from the groupconsisting of an antibody and a fragment of an antibody. Yet further,the targeted-superantigen is selected from the group consisting of, butnot limited to SEQ. ID. NO. 5, SEQ. ID. NO. 6, SEQ. ID. NO. 7, SEQ. ID.NO. 8, SEQ. ID. NO. 9, and SEQ. ID. NO. 10.

The chemotherapeutic drug can be a cytostatic drug. For example, thecytostatic drug is selected from the group consisting of, but notlimited to alkylating agents, antimetabolites, inhibitors of mitosis,anti-tumor antibiotics, and platinum based compounds.

In certain embodiments, the alkylating agent is selected from the groupconsisting of, but not limited to busulfan, chlorambucil,cyclophosphamide, melphalan, carmustine, and lomustine.

Still further, the antimetabolite can be selected from the groupconsisting of, but not limited to 5-fluorouracil, gemcitabine, andpemetrexed.

More particularly, the antitumor antibiotic is selected from the groupconsisting of, but not limited to doxorubicin, daunorubicin, mitomycin,actinomycin D, and bleomycin.

In further embodiments, the mitotic inhibitor is selected from the groupconsisting of, but not limited to paclitaxel, docetaxel, vinblastine,vincristine, and etoposide.

Still further, the platinum based compound is selected from the groupconsisting of, but not limited to cisplatin, carboplatin, andoxaliplatin.

In certain embodiments, the mammal, for example a human, is sufferingfrom a hyperproliferative disease, wherein the hyperproliferativedisease is further defined as cancer. More particularly, the cancer isselected from the group consisting of, but not limited to lung, breast,colon, kidney, pancreatic, ovarian, stomach, cervix and prostate cancer.

Another embodiment of the present invention comprises a method forenhancing the T cell response a tumor-targeted superantigen in a mammal,comprising systemically administering to the mammal a tumor-targetedsuperantigen and a chemotherapeutic agent, wherein administration of thechemotherapeutic agent reduces the antibody response to thetumor-targeted superantigen. The mammal is a human.

Still further, another embodiment of the present invention comprises amethod for increasing the anti-tumor effect of a chemotherapeutic agentin a mammal, comprising administering to the mammal a chemotherapeuticagent and systemically administering to the mammal a tumor-targetedsuperantigen. The mammal is a human.

In preferred embodiments of the present invention, the superantigen is abacterial superantigen, and the anticancer agent is a chemotherapy drug,such that the superantigen is directed to cells or other locations thatexpress the antigen recognized by the targeting moiety. In certainembodiments, the targeting moiety may be an antibody or an antibodyfragment. In certain embodiments, the targeting moiety may be a solubleT cell receptor (TCR) (e.g., a soluble TCR) or other specific bindingmoiety. In certain embodiments, the superantigen may be modified, forexample, by genetic engineering or other treatments, to have modifiedproperties from wild-type superantigen, for example but not limited to,reduced MHC class II binding and/or reduced seroreactivity and/orability to generate an antibody response.

An embodiment of the present invention is a method for treating ahyperproliferative disease in a mammal comprising the steps ofadministering to the mammal a therapeutically effective amount of asuperantigen, such as TTS, and administering a therapeutically effectiveamount of an anticancer agent. In certain embodiments, the anticanceragent is a chemotherapeutic drug or agent, and/or the anticancer agentmay be radiation. In preferred embodiments, the mammal is a human. Inother preferred embodiments, the hyperproliferative disease is cancer.In other preferred embodiments, the anticancer agent is achemotherapeutic drug.

Still further, in some embodiments of the present invention thechemotherapeutic drug is a cytostatic drug. In certain embodiments, thecytostatic drug may have a mechanism of action selected from the groupconsisting of alkylating agents (e.g., busulfan, chlorambucil,cyclophosphamide, melphalan, carmustine, and lomustine), antimetabolites(e.g., 5-fluorouracil, gemcitabine, and pemetrexed), inhibitors ofmitosis (e.g., paclitaxel, docetaxel, vinblastine, vincristine, andetoposide), anti-tumor antibiotics (e.g., doxorubicin, daunorubicin,mitomycin, actinomycin D, and bleomycin), and platinum based compounds(e.g., cisplatin, carboplatin, and oxaliplatin).

In further embodiments of the present invention, the superantigen is awild-type superantigen or a modified superantigen. The superantigen, insome embodiments of the present invention, is conjugated to at least onetargeting moiety. The targeting moiety, in certain embodiments, is anantibody moiety. More particularly, the antibody moiety may be selectedfrom the group consisting of polyclonal antibodies, monoclonalantibodies, Fv, Fab and F(ab)₂, single chain antibodies, nanobodies(Gibbs, 2005), and humanized antibodies. Still further, the targetingmoiety is a soluble T cell receptor.

In preferred embodiments of the present invention the superantigen isderived from a bacterium or a virus. More particularly, the superantigenis staphylococcal enterotoxin E, modified staphylococcal enterotoxin E,staphylococcal enterotoxin A, or modified staphylococcal enterotoxin A.Still more particularly, the superantigen is C215Fab-SEA having theamino acid sequence of SEQ ID NO 5; 5T4Fab-SEAD227A having the aminoacid sequence SEQ ID NO 6; or 5T4Fab-SEA/E-120 having the amino acidsequence of SEQ ID NO 7. Still further, the superantigen is C215Fab-SEAand is encoded by the nucleic acid sequence of SEQ ID NO 9; is5T4Fab-SEAD227A encoded by the nucleic acid sequence SEQ ID NO 8; or is5T4Fab-SEA/E-120 encoded by the nucleic acid sequence of SEQ ID NO 10.In some embodiments the superantigen has the amino acid sequenceaccording to SEQ ID NO 2.

In some embodiments of the present invention the superantigen, such asTTS is encoded by a DNA sequence that has been modified so that thesuperantigen has reduced MHC class II binding, relative to wild-typesuperantigen. In some embodiments the superantigen is encoded by a DNAsequence that has been modified so that the superantigen has reducedseroreactivity compared to wild-type superantigen.

In certain embodiments of the present invention the superantigen isadministered in an amount of from 0.001 to 500 μg/kg body weight of thesubject.

In some embodiments the superantigen is administered prior to theadministration of the anti-cancer agent. In some embodiments thesuperantigen is administered partially simultaneously with theadministration of the anti-cancer agent. In some embodiments thesuperantigen is administered simultaneous with the administration of theanti-cancer agent. Further, in some embodiments of the present inventionthe superantigen is administered subsequent to the administration of theanticancer agent.

In some embodiments of the present invention a cytostatic drug isadministered first, and a superantigen is administered when the level ofthe cytostatic drug drops to where it is no longer cytostatic. In someembodiments a cytostatic drug is administered first, and a superantigenis administered 0 to 6 days following the administration of thecytostatic agent. In certain embodiments of the invention, a cytostaticdrug is administered first, and a superantigen is administered 12-72hours following the administration of the cytostatic agent. In someembodiments a cytostatic drug is administered first, and a superantigenis administered 0 to 6 days after the level of the cytostatic drug dropsto where it is no longer cytostatic. In some embodiments of theinvention a cytostatic drug is administered first, and a superantigen isadministered 0-72 hours after the level of the cytostatic drug drops towhere it is no longer cytostatic. Further, in some embodiments acytostatic drug is administered first, and a superantigen isadministered 12-72 hours after the level of the cytostatic drug drops towhere it is no longer cytostatic.

In some embodiments of the invention a superantigen, such as TTS and ananticancer agent are administered within 0 to 6 days of each other. Insome embodiments, a superantigen and an anticancer agent areadministered within 0-72 hours of each other. In some cases, asuperantigen an an anticancer agent are administered within 3-4 monthsof each other. Optionally, a superantigen and an anticancer agent areadministered within 1 month of each other. Further, in some instanceswithin the scope of the present invention, a superantigen and ananticancer agent are administered within 2 weeks of each other. Stillfurther, in some embodiments a superantigen and an anticancer agent areadministered within 1 week of each other. Also, in some embodiments asuperantigen and an anticancer agent are administered within 7-10 daysof each other. Still further, in some cases a superantigen and ananticancer agent are administered within 0-30 days of each other.Optionally, in some embodiments a superantigen and an anticancer agentare administered within 1-10 days of each other. In other embodiments, asuperantigen and an anticancer agent are administered within 0-72 hoursof each other. In some embodiments a superantigen and an anticanceragent are administered within 0-24 hours of each other. In still otherembodiments, a superantigen and an anticancer agent are administeredwithin 0-12 hours of each other.

In certain preferred embodiments, a superantigen and an anticancer agentare administered in sequential dosage. In some embodiments of sequentialdosage administration, a superantigen is administered first. Yetfurther, in some embodiments of sequential dosage administration, aanticancer agent is administered first. In some embodiments ofsequential dosage administration the sequential dosage follows a patternselected from the following, or approximations thereof, whereA=superantigen and B=anticancer agent: A/B/A B/A/B B/B/A A/A/B A/B/BB/A/A A/B/B/B; B/A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A; B/B/A/AB/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A; and A/A/B/A.

In certain embodiments of sequential dosage administration, theadministration of a superantigen and the administration of an anticanceragent are separated by 0-6 days. In certain embodiments of sequentialdosage the administration of a superantigen and the administration of ananticancer agent are separated by 0-72 hours.

In preferred embodiments, the hyperproliferative disease is associatedwith the formation of tumors or other forms of localized concentrationsof hyperproliferative cells.

In preferred embodiments of the present invention, thehyperproliferative disease to be treated is cancer and is selected fromthe group consisting of tumors of lung, breast, colon, kidney,pancreatic, ovarian, stomach, cervix and prostate cancer. In preferredembodiments, a human is treated for a cancer selected from the groupconsisting of lung, breast, colon, kidney, pancreatic, ovarian, stomach,cervix and prostate cancer. More preferred embodiments include thetreatment of a human having tumors associated with a cancer selectedfrom the group consisting of lung, breast, colon, kidney, pancreatic,ovarian, stomach, cervix and prostate cancer, with a superantigen thatis a wild-type or modified Staphylococcal bacterial superantigen fusedto a targeting moiety. In still more preferred embodiments, a humanhaving one of the above-described cancers is treated with a superantigenselected from the group consisting of C215Fab-SEA having the amino acidsequence of SEQ ID NO 5, 5T4Fab-SEAD227A having the amino acid sequenceSEQ ID NO 6, and 5T4Fab-SEA/E-120 having the amino acid sequence of SEQID NO 7, and an anticancer agent selected from the group consisting ofgemcitabine or docetaxel.

Certain preferred embodiments of the present invention include a methodfor treating a mammal comprising the steps of administering to themammal a therapeutically effective amount of a superantigen, and atherapeutically effective amount of an anticancer agent, wherein theanticancer agent reduces formation of antibodies in the mammal to thesuperantigen and/or targeting moiety of the TTS compared with theadministration of a superantigen alone. In more preferred embodiments ofthis method, the mammal is a human and the superantigen is TTS.

Additional embodiments of the instant invention include kits comprisinga first container having a superantigen and a second container having ananti-cancer agent. Such kits, in some embodiments, are for the treatmentof a disease of a mammal, which in preferred embodiments is a human. Inmore preferred embodiments, the mammal is a human and the diseasetreated is a cancer. In still more preferred embodiments of the kit, thesuperantigen is selected from the group consisting of C215Fab-SEA havingthe amino acid sequence of SEQ ID NO 5, 5T4Fab-SEAD227A having the aminoacid sequence SEQ ID NO 6, or 5T4Fab-SEA/E-120 having the amino acidsequence of SEQ ID NO 7, and the anticancer agent is selected from thegroup consisting of gemcitabine or docetaxel. In still other embodimentsof the kit, superantigen is C215Fab-SEA having the amino acid sequenceof SEQ ID NO 5, and/or the superantigen is 5T4Fab-SEAD227A having theamino acid sequence SEQ ID NO 6 and/or the superantigen is5T4Fab-SEA/E-120 having the amino acid sequence of SEQ ID NO 7. Stillfurther, the kit comprises a superantigen, for example, C215Fab-SEAencoded by the nucleic acid sequence of SEQ ID NO 9; 5T4Fab-SEAD227Aencoded by the nucleic acid sequence SEQ ID NO 8; or 5T4Fab-SEA/E-120encoded by the nucleic acid sequence of SEQ ID NO 10.

The foregoing has outlined rather broadly the features and technicaladvantages of the present invention in order that the detaileddescription of the invention that follows may be better understood.Additional features and advantages of the invention will be describedhereinafter which form the subject of the sentences of the invention. Itshould be appreciated that the conception and specific embodimentdisclosed may be readily utilized as a basis for modifying or designingother structures for carrying out the same purposes of the presentinvention. It should also be realized that such equivalent constructionsdo not depart from the invention as set forth in the appended. The novelfeatures which are believed to be characteristic of the invention, bothas to its organization and method of operation, together with furtherobjects and advantages will be better understood from the followingdescription when considered in connection with the accompanying figures.It is to be expressly understood, however, that each of the examples andfigures are provided for the purpose of illustration and descriptiononly and are not intended as a definition of the limits of the presentinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present invention, reference isnow made to the following descriptions taken in conjunction with theaccompanying drawing, in which:

FIGS. 1A-1D show a schematic showing of exemplary embodiments of thepresent invention regarding administration of TTS and a chemotherapeuticagent to an animal, such as a human. FIG. 1A shows the administration ofa chemotherapeutic agent prior to administration of TTS. FIG. 1B showsthe administration of TTS prior to the administration of achemotherapeutic agent. FIG. 1C shows the administration of achemotherapeutic agent followed by administration of TTS followed bysimultaneous administration of TTS and a chemotherapeutic agent. FIG. 1Dshows the administration of TTS followed by administration simultaneousadministration of TTS and a chemotherapeutic agent.

FIG. 2 a schematic showing embodiments of the present inventionregarding administration of TTS and a chemotherapeutic agent to ananimal, such as a human.

FIG. 3 is a comparison of SEA, SEE and SEA/E-120 superantigens.

FIG. 4 shows that pre-treatment with docetaxel (TAXOTERE®) enhancestumor cell sensitivity for ABR-217620 induced SADCC.

FIG. 5 shows CTL activity in bulk splenocytes from mice treatedsequentially with gemcitabine at various doses and then Fab-SEA. Mice (3mice/group) were given four injections of gemcitabine at indicated dosesevery third day (days 1, 4, 7 and 10) followed by treatment with threedaily injections of C215Fab-SEA (10 μg/animal), starting at day 16. 48hours after the last injection, spleens were removed and cytotoxicfunction (SDCC) against SEA coated A20 cells, was measured in a standard⁵¹Cr release assay. The effector to target cell ratio was 100:1. Datapoints representing results from individual mice, as well as trend linesrepresenting average values from 3 individual mice.

FIGS. 6A-6D show T-cell dynamics of splenocytes of mice treatedsequentially with gemcitabine at various doses and then Fab-SEA. Mice (3animals/group) were given four injections of gemcitabine at indicateddoses every third day (days 1, 4, 7 and 10) followed by treatment withthree daily injections of C215Fab-SEA (10 μg/animal), starting at day16. 48 hours after the last injection, spleens were removed and V_(β)3specific expansion of CD4⁺ and CD8⁺ cells was measured. Data pointsrepresenting results from individual mice, as well as trend linesrepresenting average values from 3 individual. FIG. 6A shows expansionof CD4⁺ V_(β)3 T cells (SEA reactive) using various concentrations ofgemcitabine. FIG. 6B shows expansion of CD4⁺ V_(β)8 T cells (control)using various concentrations of gemcitabine. FIG. 6C shows CD8⁺ V_(β)3 Tcells (SEA reactive) using various concentrations of gemcitabine. FIG.6D shows CD8⁺ V_(β)8 T cells (control) using various concentrations ofgemcitabine.

FIGS. 7A-7B show T-cell dynamics of splenocytes from mice treatedsequentially with gemcitabine and then Fab-SEA. Mice (3 mice/group) wereinjected with gemcitabine (0, 1, 2.4 mg/mouse) every third day for four(filled diamonds) or seven doses (open diamonds), followed by treatmentwith three daily injections of C215Fab-SEA (10 μg/animal), starting 6days after the last gemcitabine injection. 48 hours after the lastinjection, spleens were removed and V_(β)3 specific expansion of CD4 andCD8 cells was measured. Data points representing results from individualmice, as well as trend lines representing average values from 3individual mice. FIG. 7A shows the number of CD4⁺ V_(β)3 T cells (SEAreactive) using various concentrations and dosing schedules ofgemcitabine. FIG. 7B shows the number of CD4⁺ V_(β)8 T cells (control)using various concentrations and dosing schedules of gemcitabine. FIG.7C shows the number of CD8⁺ V_(β)3 T cells (SEA reactive) using variousconcentrations and dosing schedules of gemcitabine. FIG. 7D shows thenumber of CD8⁺ V_(β)8 T cells (control) using various concentrations anddosing schedules of gemcitabine.

FIG. 8 shows CTL activity in bulk splenocytes from mice treatedsequentially with 4 or 7 doses of gemcitabine and then Fab-SEA. Mice (3animals/group) were injected with gemcitabine (0, 1, 2.4 mg/mouse) everythird day for four (filled diamonds) or seven doses (open diamonds),followed by treatment with three daily injections of C215Fab-SEA (10μg/animal), starting 6 days after the last gemcitabine injection. 48hours after the last injection, spleens were removed and cytotoxicfunction (SDCC) against SEA coated A20 cells was measured in a standard⁵¹Cr release assay. The effector to target cell ratio was 100:1. Datapoints representing results from individual mice, as well as trend linesrepresenting average values from 3 individual mice.

FIGS. 9A-9D show T-cell dynamics of splenocytes of mice treatedsequentially with gemcitabine and then Fab-SEA. Mice (3 mice/group) wereinjected with gemcitabine (2.4 mg/mouse) every third day for four doses(days 1, 4, 7 and 10), followed by treatment with three daily injectionsof C215Fab-SEA (10 μg/animal), starting at different time intervalsafter the last gemcitabine injection. 48 hours after the last injection,spleens were removed and V_(β)3 specific expansion of CD4 and CD8 cellswas measured. Data points representing results from individual mice, aswell as trend lines representing average values from 3 individual mice.FIG. 9 shows the number of CD4⁺ V_(β)3T cells (SEA reactive) usingvarious concentrations of gemcitabine. FIG. 9B shows the number of CD4⁺V_(β)8 T cells (control) using various concentrations of gemcitabine.FIG. 9C shows the number of CD8⁺ V_(β)3 T cells (SEA reactive) usingvarious concentrations of gemcitabine. FIG. 9D shows the number of CD8⁺V_(β)8 T cells (control) using various concentrations of gemcitabine.

FIG. 10 shows CTL activity in bulk splenocytes from mice treatedsequentially with gemcitabine and then Fab-SEA. Mice (3 mice/group) wereinjected with gemcitabine (2.4 mg/mouse) every third day for four doses(days 1, 4, 7 and 10), followed by treatment with three daily injectionsof C215Fab-SEA (10 μg/animal), starting at different time intervalsafter the last gemcitabine injection. 48 hours after the last injection,spleens were removed and cytotoxic function (SDCC) against SEA coatedA20 cells was measured in a standard ⁵¹Cr release assay. The effector totarget cell ratio was 100:1. Data points representing results fromindividual mice, as well as trend lines representing average values from3 individual mice.

FIG. 11 shows CTL activity in bulk splenocytes from mice treatedsequentially with docetaxel at various doses and then Fab-SEA. Mice (3animals/group) were given one i.p. injection of docetaxel at indicateddoses followed by treatment with three daily injections of C215Fab-SEA(10 mg/animal), starting 1 day after cytostatic treatment. 48 hoursafter the last Fab-SEA injection, spleens were removed and cytotoxicfunction (SDCC) against SEA coated A20 cells was measured in a standard⁵¹Cr release assay. The effector to target ratio was 100:1. Data pointsrepresenting results from individual mice, as well as trend linesrepresenting average values from 3 individual mice.

FIGS. 12A-12D show T-cell dynamics of splenocytes of mice treatedsequentially with docetaxel at various doses and then Fab-SEA. Mice (3animals/group) were given one i.p. injection of docetaxel at indicateddoses followed by treatment with three daily injections of C215Fab-SEA(10 μg/animal), starting 1 day after cytostatic treatment. 48 hoursafter the last Fab-SEA injection, spleens were removed and VP specificexpansion of CD4 and CD8 cells was measured. Data points representingresults from individual mice, as well as trend lines representingaverage values from 3 individual mice. FIG. 12A shows the number of CD4⁺V_(β)3 T cells (SEA reactive) using various concentrations of docetaxel.FIG. 12B shows the number of CD4⁺ V_(β)8 T cells (control) using variousconcentrations of docetaxel. FIG. 12C shows the number of CD8⁺ V_(β)3 Tcells (SEA reactive) using various concentrations of docetaxel. FIG. 12Dshows the number of CD8⁺ V_(β)8 T cells (control) using variousconcentrations of docetaxel.

DETAILED DESCRIPTION OF THE INVENTION

It is readily apparent to one skilled in the art that variousembodiments and modifications may be made to the present inventionwithout departing from the scope and spirit of the invention.

The present invention relates to methods of treating mammals, forexample humans, by administering a superantigen and an anticancer agent.The inventors have discovered that combined administration ofsuperantigens (which may be called a form of immune therapy) withanticancer agents, such as cytostatic drugs, results in enhancedanticancer effect for both agents, compared to when each agent isadministered alone. Further, this combined therapy results in a reducedantibody response to the superantigen, compared to administration of thesuperantigen alone, which assists in treatment, especially in caseswhere it is desired to administer superantigens several different timesthroughout a course of treatment.

I. DEFINITIONS

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. For purposes of the presentinvention, the following terms are defined below.

As used herein the specification, “a” or “an” may mean one or more. Asused herein in conjunction with the word “comprising”, the words “a” or“an” may mean one or more than one. As used herein “another” may mean atleast a second or more. For example, a statement such as “treatment witha superantigen and a cytostatic agent,” whether in the specification orclaims of this application may mean treatment: with one superantigen andone anticancer agent; with more than one superantigen and one anticanceragent; with one superantigen and more than one anticancer agent; withmore than one superantigen and more than one anticancer agent; or withvarious other combinations thereof.

As used herein, the term “antibody” refers to an immunoglobulinmolecule, which is able to specifically bind to a specific object, suchas, for example, an epitope on an antigen. As used herein, an antibodyis intended to refer broadly to any immunologic binding agent such asIgG, IgM, IgA, IgD and IgE, as well as synthetic and modifiedantibodies, as well as to antibodies from or derived from variousanimals, such as but not limited to, humans, mice and llamas. Antibodiescan be intact immunoglobulins derived from natural sources, or fromrecombinant sources and can be immunoactive portions of intactimmunoglobulins. The antibodies in the present invention may exist in avariety of forms including, for example, polyclonal antibodies,monoclonal antibodies, Fv, Fab and F(ab)₂, as well as single chainantibodies, chimeric antibodies, humanized antibodies, and fullyhumanized antibodies (Bird et al., 1988).

As used herein, the terms “disease,” “disorder” and “condition,”describe any condition or disease of a mammal, for example, but notlimited to a human, and are intended to have a broad coverage, coveringall types of diseases and disorders, including but not limited tocancers, neoplasms, and other types of hyperproliferative diseases anddisorders.

As used herein, the terms “agent” and “drug,” may be interchangeable.For example, where it is clear from the context, the terms “cytostaticagent(s),” or “anticancer agent(s),” may mean the same thing as theterms “cytostatic drug(s),” or “anticancer drug(s),” respectively.

As used herein, the terms “cancer” and “cancerous” refer to or describethe physiological condition in mammals that is typically characterizedby unregulated cell growth. Examples of cancer include, but are notlimited to, melanoma, carcinoma, lymphoma, blastoma, sarcoma, andleukemia or lymphoid malignancies. More particular examples of cancersinclude squamous cell cancer (e.g., epithelial squamous cell cancer),lung cancer including small-cell lung cancer, non-small cell lungcancer, adenocarcinoma of the lung and squamous carcinoma of the lung,cancer of the peritoneum, hepatocellular cancer, gastric or stomachcancer including gastrointestinal cancer, pancreatic cancer,glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladdercancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectalcancer, endometrial cancer or uterine carcinoma, salivary glandcarcinoma, kidney or renal cancer, prostate cancer, vulval cancer,thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, aswell as head and neck cancer.

As used herein, the term “hyperproliferative disease” is defined as adisease that results from a hyperproliferation of cells. Exemplaryhyperproliferative diseases include, but are not limited to, cancer orautoimmune diseases. Examples include, but are not limited to, cancers,such as the cancer is melanoma, non-small cell lung, small-cell lung,lung, hepatocarcinoma, retinoblastoma, astrocytoma, glioblastoma, gum,tongue, leukemia, neuroblastoma, head, neck, breast, pancreatic,prostate, renal, bone, testicular, ovarian, mesothelioma, cervical,gastrointestinal, lymphoma, brain, colon, sarcoma or bladder cancer. Thecancer may include a tumor comprised of tumor cells. In otherembodiments, the hyperproliferative disease is rheumatoid arthritis,inflammatory bowel disease, osteoarthritis, leiomyomas, adenomas,lipomas, hemangiomas, fibromas, vascular occlusion, restenosis,atherosclerosis, pre-neoplastic lesions (such as adenomatous hyperplasiaand prostatic intraepithelial neoplasia), carcinoma in situ, oral hairyleukoplakia, or psoriasis.

As used herein, the term “immunogen” is defined as a molecule thatprovokes (evokes, induces, or causes) an immune response. This immuneresponse may involve antibody production, the activation of certaincells, such as, for example, specific immunologically-competent cells,or both. An immunogen may be derived from many types of substances, suchas, but not limited to, molecules from organisms, such as, for example,proteins, subunits of proteins, killed or inactivated whole cells orlysates, synthetic molecules, and a wide variety of other agents bothbiological and nonbiological. Therefore, a skilled artisan realizes thatany macromolecule, including virtually all proteins, can serve asimmunogens. Furthermore, immunogens can be derived from recombinant DNA.

As used herein, the term “immunogenicity” is related to the ability ofan immunogen to provoke (evoke, induce, or cause) an immune response. Asis known in the art, different molecules may have differing degrees ofimmunogenicity, and a molecule having an immunogenicity that is greatercompared to another molecule is known, for example, to be capable ofprovoking (evoking, inducing, or causing) a greater immune response thanwould an agent having a lower immunogenicity. As is know in the art,certain agents may have no immunogenicity.

As used herein, the term “antigen” is defined as a molecule that isrecognized by antibodies, specific immunologically-competent cells, orboth. An antigen may be derived from many types of substances, such as,but not limited to, molecules from organisms, such as, for example,proteins, subunits of proteins, killed or inactivated whole cells orlysates, synthetic molecules, and a wide variety of other agents bothbiological and nonbiological. Therefore, a skilled artisan realizes thatany macromolecule, including virtually all proteins, can serve asantigens. Furthermore, antigens can be derived from recombinant DNA.

As used herein, the term “antigenicity” is related to the ability of anantigen to be recognized by antibodies, specificimmunologically-competent cells, or both.

As used herein, the term “major histocompatibility complex,” or “MHC,”is defined as a specific cluster of genes, many of which encodeevolutionarily related cell surface proteins involved in antigenpresentation, that are among the most important determinants ofhistocompatibility. Class I MHC, or MHC-I, function mainly in antigenpresentation to CD8 T lymphocytes. Class II MHC, or MHC-II, functionmainly in antigen presentation to CD4 T lymphocytes.

As used herein, the term “conjugated” or “fused” means joined, andincludes joining by any means, including but not limited to, by chemicalmeans (e.g., chemical conjugation), by recombinant gene expression(e.g., a fusion protein) and by non-covalent means, permanently ornon-permanently, including, but not limited to, the terms fusion orfused, and including joining one or more items, objects, molecules orthe like.

As used herein, the term “derived,” for example “derived from,”includes, but is not limited to, for example, wild-type moleculesderived from biological hosts such as bacteria, viruses and eukaryoticcells and organisms, and modified molecules, for example, modified bychemical means or produced in recombinant expression systems.

As used herein, the term “container,” means any containment meanswhatsoever and is not limited to any particular containment means ordevice.

As used herein, the terms “neoplasm” or “neoplastic cells” refer tocells that multiply in an abnormal manner. Neoplasms can be classifiedas either benign, histoid, malignant, mixed multicentric, organoid orunicentric.

As used herein, the term “seroreactive,” “seroreaction” or“seroreactivity” is defined as the ability of an agent, such as amolecule, to react with antibodies in the serum of a mammal, such as,but not limited to, a human. This includes reactions with all types ofantibodies, including, for example, antibodies specific for the moleculeand nonspecific antibodies that bind to the molecule, regardless ofwhether the antibodies inactivate or neutralize the agent. As is know inthe art, different agents may have different seroreactivity relative toone another, wherein an agent having a seroreactivity lower than anotherwould, for example, react with fewer antibodies and/or have a loweraffinity and/or avidity to antibodies than would an agent having ahigher seroreactivity. This may also include the ability of the agent toelicit an antibody immune response in an animal, such as a mammal, suchas a human.

As used herein, the term “soluble T cell receptor,” or “soluble TCR,”refers to a “soluble” T cell receptor consisting of the chains of afull-length (e.g., membrane bound) receptor, except that, minimally, thetransmembrane region of the receptor chains are deleted or mutated sothat the receptor, when expressed by a cell, will not associate with themembrane. Typically, a soluble receptor will consist of only theextracellular domains of the chains of the wild-type receptor (e.g.,lacks the transmembrane and cytoplasmic domains).

As used herein, the term “superantigen” is defined as a class ofmolecules that stimulate a subset of T-cells by binding to MHC class IImolecules and Vβ domains of T-cell receptors, stimulating the activationof T-cells expressing particular Vβ V gene segments. This term includeswild-type and natural superantigens, for example, those isolated fromcertain bacteria viruses or expressed from unmodified genes from same,as well as modified superantigens, wherein, for example, the DNAsequence encoding a superantigen has been modified by geneticengineering, for example, but not limited to, to produce a fusionprotein with a targeting moiety, and/or to alter certain properties ofthe superantigen, such as, but not limited to, its MHC class II binding(for example, to reduce affinity) and/or its seroreactivity, and/or itsimmunogenicity, and/or antigenicity (for example, to reduce itsseroreactivity). This definition includes synthetic molecules having theproperties of a superantigen as described herein. This definitionincludes the superantigens, including wild-type and modified, andconjugated/fused/targeted superantigens described in the following U.S.patents and patent applications, which are hereby incorporated herein byreference in their entireties: U.S. Pat. Nos. 5,858,363, 6,197,299,6,514,498, 6,713,284, 6,692,746, 6,632,640, 6,632,441, 6,447,777,6,399,332, 6,340,461, 6,338,845, 6,251,385, 6,221,351, 6,180,097,6,126,945, 6,042,837, 6,713,284, 6,632,640, 6,632,441, 5,859,207,5,728,388, 5,545,716, 5,519,114, U.S. patent application Ser. Nos.08/765,695 (filed Jul. 25, 1997), 10/283,838 (filed Oct. 20, 2002) (U.S.Application Publication No. 20030092894), 09/463,470 (filed Jan. 21,2000), and 09/900,766 (filed Jul. 6, 2001) (U.S. Application PublicationNo. 20030039655), U.S. Patent Application Nos. 20040142464, 20030157113,20030124142, 20030036644, 20030009015, 20020177551, 20020141981,20020115190, 20020086813, 20020058032, 20020051765, 20020039585,20020028211, 20020018781, 20010046501, 60/378,988, 60/389,366,60/406,697, 60/406,750, 60/415,310, 60/415,400, and 60/438,686 and PCTInternational Publication Number WO/03/094846.

As used herein, the term “targeting moiety” is defined as any structurethat is able to bind to a cell surface structure, preferable a diseasespecific structure. The targeting moiety is usually different from theVβ chain epitope which the superantigen binds and the MHC class IIepitopes to which superantigens bind. Exemplary targeting moietiescomprise, but are not limited to, antibodies, antibody fragments and thelike, soluble T-cell receptors, interleukins, hormones, and growthfactors.

As used herein, the term “tumor-targeted superantigen” (sometimesreferred to herein as “TTS”) is defined as a molecule comprising one ormore superantigens (as defined herein) joined, fused or conjugated withone or more targeting moieties (as defined herein). Non-limitingexamples of tumor-targeted superantigens include but are not limited to,C215Fab-SEA (SEQ. ID. No. 5), 5T4Fab-SEA/E-120 (SEQ. ID. NO. 7) and5T4Fab-SEA_(D227A) (SEQ. ID. NO. 6).

As used herein, the term “T-cell receptor” is defined as receptor thatis specific to T cells. This definition expressly includes theunderstanding of the term as known in the art, and includes, forexample, a receptor that consists of a disulfide-linked heterodimer ofthe highly variable α or β chains expressed at the cell membrane as acomplex with the invariant CD3 chains, and a receptor made up ofvariable γ and δ chains expressed at the cell membrane as a complex withCD3 on a subset of T-cells.

As used herein, the term “tumor” refers to a localized concentration,gathering or other organization (including but not limited tohyperproliferative cells located within a sheath (theca) or organ) ofhyperproliferating (hyperproliferative) cells, including for example butnot limited to neoplastic cells, whether malignant or benign,pre-cancerous and cancerous cells.

As used herein, the term “therapeutically effective” and “effectiveamount,” is defined as the amount of the pharmaceutical composition thatproduces at least some effect in treating a disease or a condition. Forexample, in a combination according to the invention, an effectiveamount is the amount required to inhibit the growth of cells of aneoplasm in vivo. The effective amount of active compound(s) used topractice the present invention for therapeutic treatment of neoplasms(e.g., cancer) varies depending upon the manner of administration, theage, body weight, and general health of the subject. It is within theskill in the art for an attending physician or veterinarian to determinethe appropriate amount and dosage regimen. Such amounts may be referredto as an “effective” amounts. These terms include, but are not limitedto synergistic situations such as those presented and described in theinstant invention wherein a single agent alone, such as a superantigenor an anticancer agent such as a chemotherapeutic drug, may act weaklyor not at all, but when combined with each other, for example, but notlimited to, via sequential dosage, the two or more agents act to producea synergistic result.

As used herein, the term “inhibits the growth of a neoplasm” refers tomeasurably slowing, stopping, or reversing the growth rate of theneoplasm or neoplastic cells in vitro or in vivo. Desirably, the growthrate is slowed by 20%, 30%, 50%, or 70% or more, as determined using asuitable assay for determination of cell growth rates. Typically, areversal of growth rate is accomplished by initiating or acceleratingnecrotic or apoptotic mechanisms of cell death in neoplastic cells,resulting in a shrinkage of a neoplasm.

As used herein, the term “variant,” “variants,” “modified,” “altered,”“mutated,” and the like, refer to proteins or peptides and/or otheragents and/or compounds that differ from a reference protein, peptide orother compound. Variants in this sense are described below and elsewherein the present disclosure in greater detail. For example, changes in thenucleic acid sequence of the variant may be silent, e.g., they may notalter the amino acids encoded by the nucleic acid sequence. Wherealterations are limited to silent changes of this type a variant willencode a peptide with the same amino acid sequence as the referencepeptide. Changes in the nucleic acid sequence of the variant may alterthe amino acid sequence of a peptide encoded by the reference nucleicacid sequence. Such nucleic acid changes may result in amino acidsubstitutions, additions, deletions, fusions and truncations in thepeptide encoded by the reference sequence, as discussed below.Generally, differences in amino acid sequences are limited so that thesequences of the reference and the variant are closely similar overalland, in many regions, identical. A variant and reference peptide maydiffer in amino acid sequence by one or more substitutions, additions,deletions, fusions and truncations, which may be present in anycombination. A variant may also be a fragment of a peptide of theinvention that differs from a reference peptide sequence by beingshorter than the reference sequence, such as by a terminal or internaldeletion. Another variant of a peptide of the invention also includes apeptide which retains essentially the same function or activity as suchpeptide. A variant may also be but is not limited to: (i) one in whichone or more of the amino acid residues are substituted with a conservedor non-conserved amino acid residue and such substituted amino acidresidue may or may not be one encoded by the genetic code, or (ii) onein which one or more of the amino acid residues includes a substituentgroup, or (iii) one in which the mature peptide is fused with anothercompound, such as a compound to increase the half-life of the peptide(for example, polyethylene glycol), or (iv) one in which the additionalamino acids are fused to the mature peptide, such as a leader orsecretory sequence or a sequence which is employed for purification ofthe mature peptide. Variants may be made by mutagenesis techniques,and/or altering mechanisms such as chemical alterations, fusions,adjuncts and the like, including those applied to nucleic acids, aminoacids, cells or organisms, and/or may be made by recombinant means.Variants including all of those defined above, are within the scope ofthose skilled in the art, for example, from the teachings herein andfrom the art.

As used herein, the terms “chemotherapy,” “chemotherapeutic,”“chemotherapeutic drug or agent,” “anti-cancer drug or agent,”“antiproliferative agent or drug,” “cytotoxic agent or drug,” “cytocidalagent or drug,” and “cytostatic agent or drug,” include anything, thatalone or in combination that has any affect on a hyperproliferativedisease or condition in a mammal, such as a human. These terms include,but are not limited to, chemical, biological, and physical agents thatact directly on (e.g., affects) a hyperproliferative cell (e.g., canceror tumor) or the vascularization of a hyperproliferation. In thiscontext, the term “agent” includes, but is not limited to a drug.Chemotherapeutic agents (e.g., drugs) include, but are not limited to,cytostatic agents, cytotoxic (cytocidal) agents, and anti-angiogenesisagents. The terms “chemotherapy,” “chemotherapeutic,” “chemotherapeuticdrug or agent,” “anti-cancer drug or agent,” “antiproliferative agent ordrug,” “cytotoxic agent or drug,” “cytocidal agent or drug,” and“cytostatic agent or drug,” as used herein, do not include immunemodulators (agents that act by modulating the immune system (other thanby cytotoxic or cytostatic action) such as interleukin-2 (IL-2) andlinomide.

As used herein, the term “sequential dosage” and related terminologyrefers to the administration of at least one superantigen, with at leastone anticancer agent, for example, but not limited to, achemotherapeutic drug. This definition includes staggered doses of theseagents (i.e., time-staggered) and variations in dosage amounts. Thisincludes one agent being administered before, overlapping with(partially or totally), or after administration of another agent. Thisterm generally considers the best administration scheme to achieve asynergistic combination of at least one superantigen and at least oneanticancer agent and/or to achieve administration of at least onesuperantigen while limiting or eliminating the generation of an antibodyresponse to the superantigen. Determining sequential dosageadministration plans is within the skill on one skilled in the art, fromthe background skill and teaching in the art and the teaching of thisapplication. In certain embodiments, for example, one skilled in the artwill recognize that sequential dosing, for example, with a superantigenand a cytostatic agent, depends on the half-life of the cytostatic drug.For example, as explained in detail below, dosing of a cytostatic drugand a superantigen, such as a TTS, may be calculated by firstadministering a cytostatic agent, then administering TTS at apre-determined time after the cytostatic agent, the time calculated tobe when the concentration of the cytostatic agent falls below afunctional level. By such a dosing strategy (i.e., a sequential dosage),one can achieve synergistic effects of combined superantigen andcytostatic agent administration. Such a sequenced dosage can also reducethe formation of antibodies against the superantigen in a treatedpatient compared with, for example, administration of superantigenalone, resulting in fewer anti-superantigen antibodies in a patienttreated with such a sequential dosage.

As used herein, the terms “systemic” and “systemically” refer toadministration of an agent such that the agent is exposed to at leastone system associated with the whole body, such as but not limited tothe circulatory system, immune system, and lymphatic system, rather thanonly to a localized part of the body, such as but not limited to withina tumor. Thus, for example, a systemic therapy or an agent administeredsystematically is a therapy or an agent in which at least one systemassociated with the entire body is exposed to the therapy or agent, asopposed to, rather than just a target tissue.

As used herein, the term “parenteral administration” includes any formof administration in which the compound is absorbed into the subjectwithout involving absorption via the intestines. Exemplary parenteraladministrations that are used in the present invention include, but arenot limited to intramuscular, intravenous, intraperitoneal, orintraarticular administration.

II. THE PRESENT INVENTION

FIG. 1 shows a schematic of an embodiment of the present invention. Forexample, one or more treatments of TTS are followed directly by one ormore treatments of a chemotherapeutic agent, such as a cytostatic agent.In the present invention it has, for example, been unexpectedlydiscovered that administration, such as systemic administration, of achemotherapeutic agent, such as a cytotoxic or cytostatic agent, forexample shortly following administration of TTS enhances, for examplesynergistically, the anti-tumor effect of the TTS and chemotherapeuticagent. This is unexpected as, for example, one skilled in the art at thetime of the invention understood that in order for TTS therapy to work,activation of the immune system was necessary. For example, activationof T-cells against targeted tumor cells. One skilled in the art at thetime of the invention also understood that chemotherapeutic agents suchas cytotoxic and cytostatic agents inhibit immune activation since theyinhibit cell division. Applicants have unexpectedly found thatchemotherapeutic agents, such as cytostatic and cytotoxic agents, may beadministered together with TTS with the result being an enhanced effectof both the TTS and the chemotherapeutic agents. This includes theadministration of both TTS and chemotherapeutic agents systemically, forexample, by intravenous injection. The instant invention has discoveredthat TTS and chemotherapeutic drugs may be administered in varyingcombinations and doses, including but not limited to, full doses of TTSand chemotherapeutic agents wherein the chemotherapeutic is administeredbefore TTS administration, with TTS administration or following TTSadministration, for example and including shortly following TTSadministration, as illustrated in FIG. 1. The instant invention has alsodiscovered the multiple rounds of administration of varying combinationsof TTS and chemotherapeutic agents may also be administered. Forexample, FIG. 1 shows multiple rounds of administration of TTS shortlyfollowed by administration of a chemotherapeutic agent, such as acytotoxic or cytostatic agent.

It has been discovered that combination administration of superantigensor TTS with chemotherapeutic agents reduces the development of anantibody response to the superantigen or TTS, whereas administration ofTTS alone generates an antibody response to the superantigen or TTS. Oneskilled in the art at the time of the present invention knows that whensuperantigen or TTS is administered to animals, including mammals,including humans, it is not uncommon for the animal to develop anantibody response to the superantigen or TTS (e.g., to the superantigenor targeting moiety parts of the TTS or to both) following the firstadministration. This, therefore, makes repeated TTS therapy difficult,as increasing titers of antibodies in a patient may interfere with theanti-tumor action of the superantigen or TTS therapy. It is therefore anunexpected discovery of the present invention that coadministration ofsuperantigen or TTS and chemotherapeutic agents, such as cytotoxic orcytostatic agents, for example but not limited to doses administeredsystemically, reduces the antibody response to the superantigen or TTSin a treated animal, thereby allowing for repeated administration ofsuperantigen or TTS without the problems generated by an antibodyresponse in the treated animal. One embodiment of achieving this effectis shown in the schematic treatment regime of FIG. 1, wherein achemotherapeutic agent such as a cytostatic drug, is administeredshortly following administration of each dose of TTS. It hasunexpectedly been discovered that such administration of a cytotoxicagent, for example, inhibits the generation of a B-cell/antibodyresponse to the TTS molecule, while not inhibiting—and evenenhancing—the T-cell immune response associated with TTS therapy.

FIG. 2 shows a schematic of an embodiment of the present invention. Forexample, one or more treatments of TTS are followed directly by one ormore treatments of a chemotherapeutic agent, such as a cytostatic agent.In the present invention it has, for example, been unexpectedlydiscovered that administration, such as systemic administration, of achemotherapeutic agent, such as a cytotoxic or cytostatic agent, forexample shortly following administration of TTS enhances, for examplesynergistically, the anti-tumor effect of the TTS and chemotherapeuticagent. For example, and as shown in FIG. 1B, the instant invention hasshown that a preferred administration embodiment of the presentinvention is the administration of TTS in a four (4) day cycle followedby administration of a chemotherapeutic agent on day five (5). Thisround of combination treatment may be repeated more than once, forexample and as shown in FIG. 2, by a second TTS administration on days22-25 and administration of a chemotherapeutic agent on day 26. Thiscycling of coadministration may be continued. Still further, anotherexample is shown in FIG. 1C and/or FIG. 1D in which the first TTSadministration is either before or after the administration of achemotherapeutic agent. If the chemotherapeutic agent is administeredfirst or prior to the TTS, then the TTS is started after the effectiveconcentration of the chemotherapeutic agent is reduced or has dropped inthe subject below a functional inhibitory level. This time period can beabout 24 hours or less depending upon the chemotherapeutic agent. Thus,after this time period, TTS administration may be initiated. TTS can beadministered systemically for a period of time to allow for sufficientproduction of effector cells, such time period can be about 1-2 days.After this period of time, then there can be a simultaneousadministration of the chemotherapeutic agent and TTS as shown in FIG. 1Cand FIG. 1D.

As discussed above, it has also been discovered in the present inventionthat such a combination of superantigen, such as TTS, and administrationof a chemotherapeutic drug, such as a cytotoxic drug (for example atfull dose, systemic administration), results in an unexpected inhibitionin the generation of an antibody response to the superantigen (e.g.,TTS), which, therefore, results in the unexpected ability to treat apatient, such as a human, with multiple rounds of superantigen (e.g.,TTS) while having a reduced or eliminated possibility of the patientgenerating an antibody response to the superantigen (e.g., TTS). It isunexpected that, for example, superantigen (e.g., TTS) treatment incombination with a chemotherapeutic drug, such as a cytotoxic orcytostatic drug, would inhibit the B-cell/antibody-generating responsewhile not inhibiting—and even enhancing—the T-cell/superantigen (TTS)anti-tumor response.

In illustrative examples, the TTS in FIGS. 1 and 2 may be, for examplebut not limited to, C215Fab-SEA (SEQ. ID. NO. 5) 5T4Fab-SEA/E-120 (SEQ.ID. NO. 7), or 5T4Fab-SEA_(D227A) (SEQ. ID. NO. 6) administeredsystemically, for example by intravenous administration, thechemotherapeutic agent may be a cytotoxic or cytostatic agent, forexample but not limited to, gemcitibine, docetaxel, cisplatin orpemetrexed, administered systemically, for example by intravenousadministration, and at the “full” dose (i.e., the dose normallyadministered when the agent is administered alone), and the TTS andchemotherapeutic agent may be administered to an animal, such as amammal, such as a human.

III. SUPERANTIGENS

Superantigens are bacterial proteins, viral proteins, andhuman-engineered molecules, capable of activating T lymphocytes, forexample, at picomolar concentrations. Superantigens are characterized bytheir ability to activate large subsets of T lymphocytes. They bind tothe major histocompatibility complex (MHC) without being processed.Superantigens bind to conserved regions outside the antigen-bindinggroove on MHC class II molecules, avoiding most of the polymorphism inthe conventional peptide-binding site. Superantigens bind to the T-cellreceptor (TCR) in the Vβ chain, instead of binding to the hypervariableloops of the T-cell receptor. Examples of bacterial superantigensinclude, but are not limited to, Staphylococcal enterotoxin (SE),Streptococcus pyogenes exotoxin (SPE), Staphylococcus aureus toxicshock-syndrome toxin (TSST-1), Streptococcal mitogenic exotoxin (SME),Streptococcal superantigen (SSA), Staphylococcal enterotoxin A (SEA),and Staphylococcal enterotoxin E (SEE).

The polynucleotide sequences encoding many superantigens have beenisolated and cloned and superantigens expressed from thesepolynucleotide sequences have been used in anticancer therapy.Superantigens expressed by these polynucleotide sequences may bewild-type superantigens, modified superantigens, or wild-type ormodified superantigens conjugated or fused with target-seeking moieties.Further, as explained in the following U.S. patents and patentsapplications, it is know in the art that superantigens may beadministered to a mammal, such as a human, directly, for example byinjection, or may be delivered, for example, by exposure of blood of apatient to the superantigen outside the body, or, for example, viaplacing a gene encoding a superantigen inside a mammal to be treated(e.g., via known gene therapy methods and vectors such as, for example,via cells containing, and capable of expressing, the gene) andexpressing the gene within the mammal. Other routes of administration ofsuperantigens are included within the scope of this invention.

Examples of superantigens and their administration to mammals may befound in the following U.S. patents and patent applications, each ofwhich is hereby incorporated herein by reference in its entirety: U.S.Pat. Nos. 5,858,363, 6,197,299, 6,514,498, 6,713,284, 6,692,746,6,632,640, 6,632,441, 6,447,777, 6,399,332, 6,340,461, 6,338,845,6,251,385, 6,221,351, 6,180,097, 6,126,945, 6,042,837, 6,713,284,6,632,640, 6,632,441, 5,859,207, 5,728,388, 5,545,716, 5,519,114, U.S.patent application Ser. Nos. 08/765,695 (filed Jul. 25, 1997),10/283,838 (filed Oct. 20, 2002) (U.S. Application Publication No.20030092894), 09/463,470 (filed Jan. 21, 2000), and 09/900,766 (filedJul. 6, 2001) (U.S. Application Publication No. 20030039655), U.S.Patent Application Nos. 20040142464, 20030157113, 20030124142,20030036644, 20030009015, 20020177551, 20020141981, 20020115190,20020086813, 20020058032, 20020051765, 20020039585, 20020028211,20020018781, 20010046501, 60/378,988, 60/389,366, 60/406,697,60/406,750, 60/415,310, 60/415,400, and 60/438,686 and PCT InternationalPublication Number WO/03/094846. As defined herein, the term“superantigen(s)” includes wild-type and modified superantigens as wellas targeted (e.g., conjugated or fused) superantigens. More preferably,the present invention concerns targeted (e.g., conjugated or fused)superantigens. The definition of the term “superantigen(s)” as usedherein covers any molecule(s) capable of interacting with a TCR toactivate a subset of T cells.

A. Modified Superantigens

Within the scope of this invention, superantigens may be modified fromwild-type in virtually any number of ways. Examples of preferredembodiments include modifications that retain or enhance the ability ofa superantigen to stimulate T lymphocytes, and may, for example, alterother aspects of the superantigen, such as, for example, itsseroreactivity or immunogenicity. Modified superantigens includesynthetic molecules that have superantigen activity (i.e., the abilityto activate subsets of T lymphocytes).

It is thus contemplated by the inventors that various changes may bemade in the polynucleotide sequences encoding a superantigen withoutappreciable loss of its biological utility or activity, as discussedbelow. The activity being the induction of the T-cell response to resultin cytotoxicity of the tumor cells. Yet further, the affinity of thesuperantigen for the MHC class II molecule can be decreased with minimaleffects on the cytotoxicity of the superantigen. This, for example,helps to reduce toxicity that may otherwise occur if a superantigenretains its wild-type ability to bind MHC class II antigens (as in sucha case, class II expressing cells, such as immune system cells, couldalso be affected by the response to the superantigen).

Techniques for modifying superantigens (e.g., polynucleotides andpolypeptides), including for making synthetic superantigens, are wellknown in the art and include, for example PCR mutagenesis, alaninescanning mutagenesis, and site-specific mutagenesis (U.S. Pat. Nos.5,220,007; 5,284,760; 5,354,670; 5,366,878; 5,389,514; 5,635,377; and5,789,166, each of which is incorporated herein by reference).

In some embodiments, a superantigen may be modified such that itsseroreactivity are reduced, compared to wild-type, but its ability toactivate T cells is retained or enhanced relative to wild-type. Onetechnique for making such modified superantigens is by substitutingcertain amino acids in certain regions from one superantigen to another.This is possible because many superantigens, for example but not limitedto, SEA, SEE, SED share sequence homology in certain areas that havebeen linked to certain functions (Marrack and Kappler, 1990). Forexample, in certain embodiments of the present invention, a superantigenthat has a desired T cell activation-inducing response, but anon-desired high seroreactivity, is modified such that the resultingsuperantigen retains its T cell activation ability, yet has reducedseroreactivity.

It is known and understood by those of skill in the art that the sera ofhumans normally contain various titers of antibodies againstsuperantigens. For the staphylococcal superantigens, for instance, therelative titers are TSST-1>SEB>SEC-1>SE3>SEC2>SEA>SED>SEE. As can beseen, the seroreactivity of, for example, SEE (Staphylococcalenterotoxin E) is lower than that of, for example, SEA (Staphylococcalenterotoxin A). Based on this data, one skilled in the art would preferto administer a low titer superantigen, such as, for example SEE,instead of a high titer superantigen, such as, for example, SEB(Staphylococcal enterotoxin B). However, as has also been discovered bythe present inventors, different superantigens have differing T cellactivation properties relative to one another, and for wild-typesuperantigens, the best T cell activating superantigens often also haveundesirably high seroreactivity.

One skilled in the art also realizes that these relative titers indicatepotential problems with seroreactivity, such as problems withneutralizing antibodies. Thus, the present invention contemplates usinga low titer superantigen, such as SEA or SEE to avoid the seroreactivityof parenterally administered superantigens. A “low titer superantigen”has a low seroreactivity as measured, for example, by typicalanti-superantigen antibodies in a general population. In some instancesit may also have a low immunogenicity. Such low titer superantigens maybe modified to retain its “low titer” as described herein.

The present inventors have discovered ways of modifying superantigenssuch that, for example, a modified superantigen may be created that hasboth the desired T cell activation properties and reducedseroreactivity, and in some instances also reduced immunogenicity. Oneway of accomplishing this is by the inventors' discovery that certainregions of homology between superantigens relate to seroreactivity.Using this information, it is within the skill of one in the art toengineer a recombinant superantigen that has a desired T cell activationand a desired seroreactivity and/or immunogenicity.

Yet further, it is clearly known and understood that the proteinsequences and immunological cross-reactivity of the superantigens orstaphylococcal enterotoxins are divided into two related groups. Onegroup consists of SEA, SEE and SED. The second group is SPEA, SEC andSEB. Thus, the present invention also contemplates the use of low titersuperantigens to decrease or eliminate the cross-reactivity of thepresent invention with high titer or endogenous antibodies againststaphylococcal enterotoxins.

Regions the superantigens that have been identified as playing a role inseroreactivity include, for example, Region A, comprises amino acidresidues 20, 21, 22, 23, 24, 25, 26, and 27; Region B comprises aminoacid residues 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, and 49; Region C 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, and 84;Region D comprises amino acid residues 187, 188, 189 and 190; and RegionE comprise the amino acid residues, 217, 218, 219, 220, 221, 222, 223,224, 225, 226, and 227 (U.S. patent application Ser. No. 09/900,766(filed Jul. 6, 2001) (U.S. Application Publication No. 20030039655),which are incorporated herein by reference in their entireties). Thus,it is contemplated that these identified regions are the regions inwhich one of skill in the art would mutate using, for example amino acidsubstitution, to produce a superantigen having altered seroreactivity.

Polypeptide or amino acid sequences for the above listed superantigenscan be obtained from any sequence data bank, for example Protein DataBank and/or GenBank. Exemplary GenBank accession numbers include, butare not limited to, SEE is P12993; SEA is P013163; SEB is P01552; SECTis P01553; SED is P20723; and SEH is AAA19777. Yet further, one skilledin the art can obtain the nucleic acid sequences of the above listedsuperantigens and other superantigens from GenBank.

In certain embodiments of the present invention, the wild-type SEE (SEQ.ID. NO. 1) or SEA sequence or (SEQ. ID. NO. 2) can be modified such thatamino acids in any of the identified regions A-E are substituted withSEE amino acids. Such substitutions include for example, K79, K81, K83and D227 or K79, K81, K83, K84 and D227, or, for example, K79E, K81E,K83S and D227S or K79E, K81E, K83S, K84S and D227A. More particularly,the superantigen is SEA/E-120 (SEQ. ID. NO. 3; see also U.S. patentapplication Ser. No. 09/900,766 (filed Jul. 6, 2001) (U.S. ApplicationPublication No. 20030039655), which is incorporated herein by referencein its entirety) or SEAD227A (SEQ. ID. NO. 4; see also U.S. patentapplication Ser. No. 08/765,695 (filed Jul. 25, 1997), which isincorporated herein by reference in its entirety).

1. Modified Polynucleotides and Polypeptides

The biological functional equivalent may comprise a polynucleotide thathas been engineered to contain distinct sequences while at the same timeretaining the capacity to encode the “wild-type” or standard protein.This can be accomplished to the degeneracy of the genetic code, i.e.,the presence of multiple codons, which encode for the same amino acids.In one example, one of skill in the art may wish to introduce arestriction enzyme recognition sequence into a polynucleotide while notdisturbing the ability of that polynucleotide to encode a protein.

In another example, a polynucleotide made be (and encode) a biologicalfunctional equivalent with more significant changes. Certain amino acidsmay be substituted for other amino acids in a protein structure withoutappreciable loss of interactive binding capacity with structures suchas, for example, antigen-binding regions of antibodies, binding sites onsubstrate molecules, receptors, and such like. So-called “conservative”changes do not disrupt the biological activity of the protein, as thestructural change is not one that impinges of the protein's ability tocarry out its designed function. It is thus contemplated by theinventors that various changes may be made in the sequence of genes andproteins disclosed herein, while still fulfilling the goals of thepresent invention.

In terms of functional equivalents, it is well understood by the skilledartisan that, inherent in the definition of a “biologically functionalequivalent” protein and/or polynucleotide, is the concept that there isa limit to the number of changes that may be made within a definedportion of the molecule while retaining a molecule with an acceptablelevel of equivalent biological activity. Biologically functionalequivalents are thus defined herein as those proteins (andpolynucleotides) in selected amino acids (or codons) may be substituted.Functional activity being the induction of the T-cell response to resultin cytotoxicity of the tumor cells. Yet further, the affinity of thesuperantigen for the MHC class II molecule is decreased with minimaleffects on the cytotoxicity of the superantigen.

In general, the shorter the length of the molecule, the fewer changesthat can be made within the molecule while retaining function. Longerdomains may have an intermediate number of changes. The full-lengthprotein will have the most tolerance for a larger number of changes.However, it must be appreciated that certain molecules or domains thatare highly dependent upon their structure may tolerate little or nomodification.

Amino acid substitutions are generally based on the relative similarityof the amino acid side-chain substituents, for example, theirhydrophobicity, hydrophilicity, charge, size, and/or the like. Ananalysis of the size, shape and/or type of the amino acid side-chainsubstituents reveals that arginine, lysine and/or histidine are allpositively charged residues; that alanine, glycine and/or serine are alla similar size; and/or that phenylalanine, tryptophan and/or tyrosineall have a generally similar shape. Therefore, based upon theseconsiderations, arginine, lysine and/or histidine; alanine, glycineand/or serine; and/or phenylalanine, tryptophan and/or tyrosine; aredefined herein as biologically functional equivalents.

To effect more quantitative changes, the hydropathic index of aminoacids may be considered. Each amino acid has been assigned a hydropathicindex on the basis of their hydrophobicity and/or chargecharacteristics, these are: isoleucine (+4.5); valine (+4.2); leucine(+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine(+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and/or arginine (−4.5).

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is generally understood inthe art (Kyte & Doolittle, 1982, incorporated herein by reference). Itis known that certain amino acids may be substituted for other aminoacids having a similar hydropathic index and/or score and/or stillretain a similar biological activity. In making changes based upon thehydropathic index, the substitution of amino acids whose hydropathicindices are within ±2 is preferred, those which are within ±1 areparticularly preferred, and/or those within ±0.5 are even moreparticularly preferred.

It also is understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity,particularly where the biological functional equivalent protein and/orpeptide thereby created is intended for use in immunologicalembodiments, as in certain embodiments of the present invention. U.S.Pat. No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with itsseroreactivity and/or antigenicity, i.e., with a biological property ofthe protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). In makingchanges based upon similar hydrophilicity values, the substitution ofamino acids whose hydrophilicity values are within ±2 is preferred,those which are within ±1 are particularly preferred, and/or thosewithin ±0.5 are even more particularly preferred.

2. Altered Amino Acids

The present invention, in many aspects, relies on the synthesis ofpeptides and polypeptides in cyto, via transcription and translation ofappropriate polynucleotides. These peptides and polypeptides willinclude the twenty “natural” amino acids, and post-translationalmodifications thereof. However, in vitro peptide synthesis permits theuse of modified and/or unusual amino acids. A table (Table 1) ofexemplary, but not limiting, modified and/or unusual amino acids isprovided herein below.

TABLE 1 Modified and/or Unusual Amino Acids Abbr. Amino Acid Abbr. AminoAcid Aad 2-Aminoadipic acid EtAsn N-Ethylasparagine BAad 3-Aminoadipicacid Hyl Hydroxylysine BAla beta-alanine, AHyl allo-Hydroxylysinebeta-Amino-propionic acid Abu 2-Aminobutyric acid 3Hyp 3-Hydroxyproline4Abu 4-Aminobutyric acid, 4Hyp 4-Hydroxyproline piperidinic acid Acp6-Aminocaproic acid Ide Isodesmosine Ahe 2-Aminoheptanoic acid Aileallo-Isoleucine Aib 2-Aminoisobutyric acid MeGly N-Methylglycine,sarcosine BAib 3-Aminoisobutyric acid MeIle N-Methylisoleucine Apm2-Aminopimelic acid MeLys 6-N-Methyllysine Dbu 2,4-Diaminobutyric acidMeVal N-Methylvaline Des Desmosine Nva Norvaline Dpm 2,2′-Diaminopimelicacid Nle Norleucine Dpr 2,3-Diaminopropionic acid Orn Ornithine EtGlyN-Ethylglycine

3. Mimetics

In addition to the biological functional equivalents discussed above,the present inventors also contemplate that structurally similarcompounds may be formulated to mimic the key portions of peptide orpolypeptides of the present invention. Such compounds, which may betermed peptidomimetics, may be used in the same manner as the peptidesof the invention and, hence, also are functional equivalents.

Certain mimetics that mimic elements of protein secondary and tertiarystructure are described in Johnson et al. (1993). The underlyingrationale behind the use of peptide mimetics is that the peptidebackbone of proteins exists chiefly to orient amino acid side chains insuch a way as to facilitate molecular interactions, such as those ofantibody and/or antigen. A peptide mimetic is thus designed to permitmolecular interactions similar to the natural molecule.

Some successful applications of the peptide mimetic concept have focusedon mimetics of β-turns within proteins, which are known to be highlyantigenic. Likely β turn structure within a polypeptide can be predictedby computer-based algorithms, as discussed herein. Once the componentamino acids of the turn are determined, mimetics can be constructed toachieve a similar spatial orientation of the essential elements of theamino acid side chains.

Other approaches have focused on the use of small,multidisulfide-containing proteins as attractive structural templatesfor producing biologically active conformations that mimic the bindingsites of large proteins Vita et al. (1995). A structural motif thatappears to be evolutionarily conserved in certain toxins is small (30-40amino acids), stable, and high permissive for mutation. This motif iscomposed of a beta sheet and an alpha helix bridged in the interior coreby three disulfides.

Beta II turns have been mimicked successfully using cyclicL-pentapeptides and those with D-amino acids. Weisshoff et al. (1999).Also, Johannesson et al. (1999) report on bicyclic tripeptides withreverse turn inducing properties.

Methods for generating specific structures have been disclosed in theart. For example, alpha-helix mimetics are disclosed in U.S. Pat. Nos.5,446,128; 5,710,245; 5,840,833; and 5,859,184. Theses structures renderthe peptide or protein more thermally stable, also increase resistanceto proteolytic degradation. Six, seven, eleven, twelve, thirteen andfourteen membered ring structures are disclosed.

Methods for generating conformationally restricted beta turns and betabulges are described, for example, in U.S. Pat. Nos. 5,440,013;5,618,914; and 5,670,155. Beta-turns permit changed side substituentswithout having changes in corresponding backbone conformation, and haveappropriate termini for incorporation into peptides by standardsynthesis procedures. Other types of mimetic turns include reverse andgamma turns. Reverse turn mimetics are disclosed in U.S. Pat. Nos.5,475,085 and 5,929,237, and gamma turn mimetics are described in U.S.Pat. Nos. 5,672,681 and 5,674,976.

Other proteins and molecules that are within the scope of this inventioninclude that that may vary in, for example, glycosylation, but retainthe same function (e.g., so-called “biosimilar” or “bioequivalent”proteins).

4. Domain Switching

Yet further, a modified superantigen can be created by substitutinghomologous regions of various proteins. This is known, in certaincontexts, as “domain switching.” Domain switching involves thegeneration of chimeric molecules using different but, in this case,related polypeptides. By comparing various superantigen proteins, onecan make predictions as to the functionally significant regions of thesemolecules. It is possible, then, to switch related domains of thesemolecules in an effort to determine the criticality of these regions tosuperantigen function. These molecules may have additional value in thatthese “chimeras” can be distinguished from natural molecules, whilepossibly providing the same function.

B. Targeted Superantigens

In certain embodiments of the present invention, a targeting moiety, forexample an antibody or antibody fragment, may be conjugated to asuperantigen, providing a targeted superantigen. If the antibody, orantibody fragment recognizes a tumor-associated antigen, the targetedsuperantigen may be called a tumor-targeted superantigen (“TTS”).Targeted superantigens retain the ability to activate large number of Tlymphocytes, and add the ability to direct the activated lymphocytes tocells bearing the target moiety. For example, TTS molecules active largenumbers of T cells and direct them to tissues containing thetumor-associate antigen bound by the targeting moiety. In suchsituations, specific target cells are killed, leaving the rest of thebody relatively unharmed. Such “magic bullet” therapy is quite desiredin the art, as non-specific anticancer agents, such as cytostaticchemotherapeutic drugs, are nonspecific and kill large numbers of cellsthat are not associated with tumors to be treated. For example, studieswith TTS have shown that inflammation by cytotoxic T lymphocytes (CTLs)into tumor tissue increases rapidly in response to the first injectionof a targeted superantigen (Dohlsten et al., 1995). This inflammationwith infiltration of CTLs into the tumor is one of the major effectorsof the anti-tumor therapeutic of targeted superantigens.

As used in the present invention, tumor-targeted superantigens (TTS)represent an immunotherapy against cancer and are therapeutic fusionproteins containing a targeting moiety and a superantigen (Dohlsten etal., 1991; Dohlsten et al., 1994). These types of compounds aredisclosed and thoroughly described in e.g., WO9201470, EP 610179, U.S.Pat. No. 5,858,363, U.S. Pat. No. 6,197,299, WO9601650, EP 766566, andWO03002143, each of which is incorporated herein by reference in itsentirety. Examples of TTS that can be used in the present inventioninclude C215Fab-SEA (SEQ. ID. NO. 5), 5T4Fab-SEA_(D227A) (SEQ. ID. NO.6) and 5T4Fab-SEA/E-120 (SEQ. ID. NO. 7).

The targeting moiety can in principle be any structure that is able tobind to a cell surface structure, preferably a disease specificstructure. The structure against which the targeting moiety is directedis usually different from (a) the Vβ chain epitope to which Superantigenbinds, and (b) the MHC class II epitopes to which superantigens bind.The target-seeking moiety is primarily selected among antibodies,including antigen binding fragments of antibodies and related entities,soluble T cell receptors, growth factors, interleukins (e.g.,interleukin-2), hormones, etc. See for example U.S. Pat. Nos. 6,080,840,6,514,498; 5,858,363; 6,197,299; U.S. Application No. US2004/0126379,US2003/0175212, US2003/0144474, US2002/0142389, US2002/119149 andInternational Publications WO2003020763, WO2004050705, WO2003002143,WO9960120 and WO9960119 each of which is incorporated herein byreference.

In certain embodiments, the targeting moiety is an antibody (e.g., Fab,F(ab)₂, Fv, single chain antibody, etc.). One of skill in the art isaware that antibodies are extremely versatile and useful cell-specifictargeting moieties because they can be generated against any cellsurface antigen of interest. Monoclonal antibodies have been generatedagainst cell surface receptors, tumor-associated antigens, and leukocytelineage-specific markers such as CD antigens. Antibody variable regiongenes can be readily isolated from hybridoma cells by methods well knownin the art. Exemplary tumor associated antigens that can be used toproduce a target moiety can include, but are not limited to gp100,Melan-A/MART, MAGE-A, MAGE (melanoma antigen E), MAGE-3, MAGE-4, MAGEA3,tyrosinase, TRP2, NY-ESO-1, CEA (carcinoembryonic antigen), PSA, p53,Mammaglobin-A, Survivin, Muc1 (mucin1)/DF3, metallopanstimulin-1(MPS-1), Cytochrome P450 isoform 1B1, 90K/Mac-2 binding protein, Ep-CAM(MK-1), HSP-70, hTERT (TRT), LEA, LAGE-1/CAMEL, TAGE-1, GAGE, 5T4, gp70,SCP-1, c-myc, cyclin B1, MDM2, p62, Koc, IMP1, RCAS1, TA90, OA1, CT-7,HOM-MEL-40/SSX-2, SSX-1, SSX-4, HOM-TES-14/SCP-1, HOM-TES-85, HDAC5,MBD2, TRIP4, NY-CO-45, KNSL6, HIP1R, Seb4D, KIAA1416, IMP1, 90K/Mac-2binding protein, MDM2, NY/ESO, and LMNA.

As used herein, exemplary cancer antibodies can include, but are notlimited to anti-CD19, anti-CD20, anti-5T4, anti-Ep-CAM, anti-Her-2/neu,anti-EGFR, anti-CEA, anti-prostate specific membrane antigen(PSMA)/folate hydrolase 1 (FOLH1), anti-IGF-1R.

Still further, the superantigen of the present invention can be fusedand/or conjugated to antibody active fragments such as C215Fab, 5T4Fab(WO8907947, which is incorporated herein by reference in its entirety)or C242Fab (Lindholm et al., WO9301303, which is incorporated herein byreference in its entirety).

Another type of targeting moiety includes a soluble T cell receptor(TCR). Some forms of soluble TCR may contain either only extracellulardomains or extracellular and cytoplasmic domains. Other modifications ofthe TCR may also be envisioned to produce a soluble TCR in which thetransmembrane domains have been deleted and/or altered such that the TCRis not membrane bound as described in U.S. Publication Application Nos.US2002/119149, US2002/0142389, US2003/0144474, and US2003/0175212, andInternational Publication Nos. WO2003020763; WO9960120 and WO9960119,each of which is incorporated herein in its entirety.

The targeting moiety can be added to the superantigen by using eitherrecombinant techniques or chemically linking of the targeting moiety tothe superantigen. These methods are well recognized for the ordinaryskilled worker and comprise a large number of variants.

1. Recombinant Linkage

For recombinant superantigens, in the form of fusion proteins or in somecases as conjugates, the obtained substance will be uniform with respectto the linking position. Either the amino terminal of a modifiedsuperantigen is linked to the carboxy terminal of the targeting moietyor vice versa. For antibodies, either the light or the heavy chain maybe utilized for fusion. For example, for Fab fragments the aminoterminal of the modified superantigen is linked to the first constantdomain of the heavy antibody chain (CH1). Yet further, the modifiedsuperantigen can also be linked to an Fab fragment by linking the lightchain or to the VH1 and VL domain to the superantigen. Still further, abridge or peptide linker can be used to fuse the to compositionstogether. Conjugation or fusion using a linker or bridge can beperformed using recombinant techniques. In such cases oligopeptidebridges containing hydrophilic amino acid residues, such as Gln, Ser,Gly, Glu, Pro, His and Arg are preferred. Particularly preferred bridgesare peptide bridges consisting of 1-10 amino acid residues, moreparticularly, for 3-7 amino acid residues. An exemplary bridge is thetripeptide GlyGlyPro.

2. Chemical Linkage

It is also envisioned that the modified superantigen may be linked tothe targeting moiety via chemical linkage. Chemical linkage of the twocompounds may require a linking or peptide bridge. The bridge ispreferably hydrophilic and exhibits one or more structure(s) selectedamong amide, thioether, disulphide etc. (See U.S. Pat. Nos. 5,858,363,6,197,299, and 6,514,498, which are incorporated herein by reference intheir entireties).

Chemical linking of a modified superantigen to a targeting moiety oftenutilizes functional groups (e.g., primary amino groups or carboxygroups) that are present in many positions in the compounds. It followsthat the final product will contain a mixture of conjugate moleculesdiffering in linking positions, as well as hetero- and homo-conjugates.

C. Expression of the Superantigens

The present invention also involves the use of expression vectors andhost cells. These expression vectors, which have been geneticallyengineered to contain the nucleic acid sequence of the superantigen, areintroduced or transformed into host cells to produce the superantigen ofthe present invention (See Dohlsten et al., 1994, Forsberg et al., 1997,Erlandsson et al., 2003 and WO2003002143, each of which is incorporatedherein by reference

Host cells can be genetically engineered to incorporate nucleic acidsequences and express peptides of the present invention. Introduction ofnucleic acid sequences into the host cell can be affected by calciumphosphate transfection, DEAE-dextran mediated transfection,transvection, microinjection, cationic lipid-mediated transfection,electroporation, transduction, scrape loading, ballistic introduction,infection or other methods. Such methods are described in many standardlaboratory manuals, such as, et al., BASIC METHODS IN MOLECULAR BIOLOGY,(1986) and Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2ndEd., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1989), each of which are incorporated herein by reference.

Representative examples of appropriate host cells include bacterialcells, such as streptococci, staphylococci, E. coli, streptomyces andBacillus subtilis cells; fungal cells, such as yeast cells andaspergillus cells; insect cells such as Drosophila S2 and Spodopteracells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 andBowes melanoma cells.

Examples of production systems for superantigens are found, for example,in U.S. patent application Ser. No. 09/463,470 (filed Jan. 21, 2000),which is incorporated herein by reference in its entirety.

D. Purification of Proteins

It will be desirable to purify the superantigen or variants thereof.Protein purification techniques are well known to those of skill in theart. These techniques involve, at one level, the crude fractionation ofthe cellular milieu to peptide and non-peptide fractions. Havingseparated the protein from other proteins, the protein of interest maybe further purified using chromatographic and electrophoretic techniquesto achieve partial or complete purification (or purification tohomogeneity). Analytical methods particularly suited to the preparationof a pure peptide are ion-exchange chromatography, size exclusionchromatography; affinity chromatography; polyacrylamide gelelectrophoresis; isoelectric focusing. A particularly efficient methodof purifying peptides is fast protein liquid chromatography or evenHPLC.

Certain aspects of the present invention concern the purification, andin particular embodiments, the substantial purification, of an encodedprotein or peptide. The term “purified protein or peptide” as usedherein, is intended to refer to a composition, isolatable from othercomponents, wherein the protein or peptide is purified to any degreerelative to its naturally-obtainable state. A purified protein orpeptide therefore also refers to a protein or peptide, free from theenvironment in which it may naturally occur.

Generally, “purified” will refer to a protein or peptide compositionthat has been subjected to fractionation to remove various othercomponents, and which composition substantially retains its expressedbiological activity. Where the term “substantially purified” is used,this designation will refer to a composition in which the protein orpeptide forms the major component of the composition, such asconstituting about 50%, about 60%, about 70%, about 80%, about 90%,about 95% or more of the proteins in the composition.

Various methods for quantifying the degree of purification of theprotein or peptide will be known to those of skill in the art in lightof the present disclosure. These include, for example, determining thespecific activity of an active fraction, or assessing the amount ofpolypeptides within a fraction by SDS/PAGE analysis. A preferred methodfor assessing the purity of a fraction is to calculate the specificactivity of the fraction, to compare it to the specific activity of theinitial extract, and to thus calculate the degree of purity, hereinassessed by a “-fold purification number.” The actual units used torepresent the amount of activity will, of course, be dependent upon theparticular assay technique chosen to follow the purification and whetheror not the expressed protein or peptide exhibits a detectable activity.

Various techniques suitable for use in protein purification will be wellknown to those of skill in the art. These include, for example,precipitation with ammonium sulphate, PEG, antibodies and the like or byheat denaturation, followed by centrifugation; chromatography steps suchas ion exchange, gel filtration, reverse phase, hydroxylapatite andaffinity chromatography; isoelectric focusing; gel electrophoresis; andcombinations of such and other techniques. As is generally known in theart, it is believed that the order of conducting the variouspurification steps may be changed, or that certain steps may be omitted,and still result in a suitable method for the preparation of asubstantially purified protein or peptide.

It is known that the migration of a polypeptide can vary, sometimessignificantly, with different conditions of SDS/PAGE (Capaldi et al.,1977). It will therefore be appreciated that under differingelectrophoresis conditions, the apparent molecular weights of purifiedor partially purified expression products may vary.

High Performance Liquid Chromatography (HPLC) is characterized by a veryrapid separation with extraordinary resolution of peaks. This isachieved by the use of very fine particles and high pressure to maintainan adequate flow rate. Separation can be accomplished in a matter ofminutes, or at most an hour. Moreover, only a very small volume of thesample is needed because the particles are so small and close-packedthat the void volume is a very small fraction of the bed volume. Also,the concentration of the sample need not be very great because the bandsare so narrow that there is very little dilution of the sample.

Gel chromatography, or molecular sieve chromatography, is a special typeof partition chromatography that is based on molecular size. The theorybehind gel chromatography is that the column, which is prepared withtiny particles of an inert substance that contain small pores, separateslarger molecules from smaller molecules as they pass through or aroundthe pores, depending on their size. As long as the material of which theparticles are made does not adsorb the molecules, the sole factordetermining rate of flow is the size. Hence, molecules are eluted fromthe column in decreasing size, so long as the shape is relativelyconstant. Gel chromatography is unsurpassed for separating molecules ofdifferent size because separation is independent of all other factorssuch as pH, ionic strength, temperature, etc. There also is virtually noadsorption, less zone spreading and the elution volume is related in asimple matter to molecular weight.

Affinity Chromatography is a chromatographic procedure that relies onthe specific affinity between a substance to be isolated and a moleculethat it can specifically bind to. This is a receptor-ligand typeinteraction. The column material is synthesized by covalently couplingone of the binding partners to an insoluble matrix. The column materialis then able to specifically adsorb the substance from the solution.Elution occurs by changing the conditions to those in which binding willnot occur (alter pH, ionic strength, temperature, etc.).

IV. ANTICANCER AGENT AND/OR THERAPY

An “anticancer” agent and/or therapy is capable of negatively affectingcancer in a subject, for example, by killing cancer cells, inducingapoptosis in cancer cells, reducing the growth rate of cancer cells,reducing the incidence or number of metastases, reducing tumor size,inhibiting tumor growth, reducing the blood supply to a tumor or cancercells, promoting an immune response against cancer cells or a tumor,preventing or inhibiting the progression of cancer, or increasing thelifespan of a subject with cancer.

Tumor cell resistance to chemotherapy and radiotherapy agents representsa major problem in clinical oncology. One goal of current cancerresearch is to find ways to improve the efficacy of chemo- andradiotherapy by combining it with an immunotherapy, such as asuperantigen. Thus, in the context of the present invention, it iscontemplated that the superantigen therapy could be used in combinationwith chemotherapeutic agents and/or radiotherapeutic agents.

A. Chemotherapeutic Agents

Chemotherapeutic drugs or agents include cytotoxic and cytostatic drugsthat alone or in combination that have an affect on a hyperproliferativedisease or condition in a mammal, such as a human. These terms include,but are not limited to, chemical, biological, and physical agents thatact directly on (e.g., affects) a hyperproliferative cell (e.g, canceror tumor) or the vascularization of a hyperproliferation. In thiscontext, the term “agent” includes, but is not limited to a drug. Theseinclude, but are not limited to cytostatic agents, cytotoxic (cytocidal)agents, and anti-angiogenesis agents, but do not include immunemodulators (agents that act by modulating the immune system (other thanby cytotoxic or cytostatic action) such as interleukin-2 (IL-2) andlinomide.

In some embodiments, a chemotherapeutic agent is used to connote acompound or composition that is administered in the treatment of cancer.Such agents or drugs may be categorized by their mode of activity withina cell, for example, whether and at what stage they affect the cellcycle. Alternatively, an agent may be characterized based on its abilityto directly cross-link DNA, to intercalate into DNA, or to inducechromosomal and mitotic aberrations by affecting nucleic acid synthesis.

Cytostatic agents are defined as agents or drugs that prevent the growthand proliferation of cells. One of skill in the art understands that acytostatic agent is a chemotherapeutic agent. More particularly, thecytostatic agents that may be used in combination with a superantigeninclude, but are not limited to, alkylating agents (e.g.,cyclophosphamide, chlorambucil, melphalan); antimetabolites (e.g.,mercaptopurine, cladribine, cytarabine, fluorouracil gemcitabine);anti-tumor antibiotics (e.g., doxorubicin, epirubicin, mitoxantrone,mitomycin); inhibitor of mitosis (e.g., vinblastine, vincristine,vinorelbine, paclitaxel, docetaxel, etoposide, topotecan, irinotecan);platinum based compounds (e.g., cisplatin, carboplatin), andcorticosteroid hormones. It is also within the scope of the invention tocombine cytocidal (cytotoxic) chemotherapeutic agents or drugs withsuperantigen administration, as well as to combine agents thatnegatively affect the vascularization of tumors (anti-angiogenesisagents) with drugs with superantigen administration.

1. Corticosteroid Hormones

Corticosteroid hormones are useful in treating some types of cancer(lymphoma, leukemias, and multiple myeloma). Though these hormones havebeen used in the treatment of many non-cancer conditions, they areconsidered chemotherapy drugs when they are implemented to kill or slowthe growth of cancer cells. Corticosteroid hormones can increase theeffectiveness of other chemotherapy agents, and consequently, they arefrequently used in combination treatments, including with superantigentherapy. Prednisone and dexamethasone are examples of corticosteroidhormones.

2. Alkylating Agents

Alkylating agents are drugs that directly interact with genomic DNA toprevent the cancer cell from proliferating. This category of cytostaticdrugs represents agents that affect all phases of the cell cycle, thatis, they are not phase-specific. More specifically, alkylating agents,or their reactive intermediates, form covalent bonds withdeoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein to forman adduct in which a methyl or ethyl group is added. DNA adducts arebelieved to play a major role in mutagenesis and clastogenesis, as wellas in carcinogenesis. DNA adducts are formed at a number of reactivesites on nucleotide bases. Common locations include the N-7 and O-6 ofguanine which are shown to be associated with mutagenesis andcarcinogenesis. In general, it seems that alkylating agents that are notparticularly ionic in nature are localized more on the ring nitrogenatoms, whereas those that have greater ionic character show greaterpreferences for reaction at the oxygen atoms in DNA. Alkylating agentscan be implemented to treat chronic leukemia, non-Hodgkin's lymphoma,Hodgkin's disease, multiple myeloma, and particular cancers of thebreast, lung, and ovary. Exemplary alkylating agents include, Busulfan(Myleran), Chlorambucil, Cyclophosphamide (Cytoxan), Dacarbazine(DTIC-Dome), Estramustine Phosphate, Ifosphamide, Mechlorethamine(Nitrogen Mustard), Melphalan (Phenylalanine Mustard), Procarbazine,Thiotepa, and Uracil Mustard.

Yet further, nitrosoureas appear to function as alkylating agents, aswell as through other mechanisms such as carbamoylation, which is areaction occurring between an isocyanate and a reactant capable oflosing a proton and involving the formation of a covalent bound betweenthe isocyanate and its reactant. Alkylation is a reaction attributed tonucleic acid alkylation and carbamoylation is attributed to proteincarbamoylation.

The nitrosoureas are converted non enzymatically into a carbonium ionand an isothiocyanate molecule. The carbonium ion acts as a typicalalkylating agent and is probably responsible for the cytotoxic action ofthe nitrosoureas. The isothiocyanate may interact with proteins andaccount for some of the toxic effects of these drugs. Nitrosoureas arehighly lipophilic, which allows them to readily cross lipophilicmembranes such as those found in the central nervous system and skin.Exemplary nitrosoureas include, Carmustine (BCNU), Lomustine (CCNU),Semustine (methyl-CCNU), and Streptozocin

A superantigen can be used to treat a cancer in combination with any oneor more of these alkylating agents, some of which are discussed below.

a) Busulfan

Busulfan (also known as myleran) is a bifunctional alkylating agent.Busulfan is known chemically as 1,4-butanediol dimethanesulfonate.

Busulfan is not a structural analog of the nitrogen mustards. Busulfanis available in tablet form for oral administration. Each scored tabletcontains 2 mg busulfan and the inactive ingredients magnesium stearateand sodium chloride. The half-life of busulfan is about 2.5 hours.Busulfan is rapidly and probably completely absorbed from the GI tract,and measurable blood concentrations are obtained within 0.5-2 hoursafter oral administration of the drug. Busulfan is slowly excreted inurine, as metabolites. About 10-50% of a dose is excreted as metaboliteswithin 24 hours.

Busulfan is indicated for the palliative treatment of chronicmyelogenous (myeloid, myelocytic, granulocytic) leukemia. Although notcurative, busulfan reduces the total granulocyte mass, relieves symptomsof the disease, and improves the clinical state of the patient.Approximately 90% of adults with previously untreated chronicmyelogenous leukemia will obtain hematologic remission with regressionor stabilization of organomegaly following the use of busulfan. It hasbeen shown to be superior to splenic irradiation with respect tosurvival times and maintenance of hemoglobin levels, and to beequivalent to irradiation at controlling splenomegaly.

In practicing the present invention, one skilled in the art would knowthat superantigen therapy could be sequentially administered incombination with busulfan, for example, by first treating with busulfan.Once the effective cytostatic concentration of busulfan has dropped inthe patient below a functional inhibitory level, the superantigen can beadministered to the patient.

b) Chlorambucil

Chlorambucil (also known as leukeran) is a bifunctional alkylating agentof the nitrogen mustard type that has been found active against selectedhuman neoplastic diseases. Chlorambucil is known chemically as4-[bis(2-chlorethyl)amino]benzenebutanoic acid.

Chlorambucil is available in tablet form for oral administration. It israpidly and completely absorbed from the gastrointestinal tract. Aftersingle oral doses of 0.6-1.2 mg/kg, peak plasma chlorambucil levels arereached within one hour and the terminal half-life of the parent drug isestimated at 1.5 hours. 0.1 to 0.2 mg/kg/day or 3 to 6 mg/m2/day oralternatively 0.4 mg/kg may be used for antineoplastic treatment.Treatment regimes are well know to those of skill in the art and can befound in the “Physicians Desk Reference” and in “Remington'sPharmaceutical Sciences” referenced herein.

Chlorambucil is indicated in the treatment of chronic lymphatic(lymphocytic) leukemia, malignant lymphomas including lymphosarcoma,giant follicular lymphoma and Hodgkin's disease. It is not curative inany of these disorders but may produce clinically useful palliation.

In practicing the present invention, one skilled in the art would knowthat superantigen therapy could be sequentially administered incombination with chlorambucil, for example, by first treating withchlorambucil. Once the effective cytostatic concentration ofchlorambucil has dropped in the patient below a functional inhibitorylevel, the superantigen can be administered to the patient.

c) Cyclophosphamide

Cyclophosphamide is 2H-1,3,2-Oxazaphosphorin-2-amine,N,N-bis(2-chloroethyl)tetrahydro-, 2-oxide, monohydrate; termed Cytoxanavailable from Mead Johnson; and Neosar available from Adria.Cyclophosphamide is prepared by condensing 3-amino-1-propanol withN,N-bis(2-chlorethyl)phosphoramidic dichloride[(ClCH₂CH₂)₂N—POCl₂] indioxane solution under the catalytic influence of triethylamine. Thecondensation is double, involving both the hydroxyl and the aminogroups, thus effecting the cyclization.

Unlike other β-chloroethylamino alkylators, it does not cyclize readilyto the active ethyleneimonium form until activated by hepatic enzymes.Thus, the substance is stable in the gastrointestinal tract, toleratedwell and effective by the oral and parental routes and does not causelocal vesication, necrosis, phlebitis or even pain. Cyclophosphamide hasa half-life of about 4-8 hours and is metabolized by the liver into itsactive components: acrolein, 4-aldophosphamide,4-hydroperoxycyclophosphamide, and nor-nitrogen mustard.

Suitable doses for adults include, orally, 1 to 5 mg/kg/day (usually incombination), depending upon gastrointestinal tolerance; or 1 to 2mg/kg/day; intravenously, initially 40 to 50 mg/kg in divided doses overa period of 2 to 5 days or 10 to 15 mg/kg every 7 to 10 days or 3 to 5mg/kg twice a week or 1.5 to 3 mg/kg/day. A dose 250 mg/kg/day may beadministered as an antineoplastic. Because of gastrointestinal adverseeffects, the intravenous route is preferred for loading. Duringmaintenance, a leukocyte count of 3000 to 4000/mm³ usually is desired.The drug also sometimes is administered intramuscularly, by infiltrationor into body cavities. It is available in dosage forms for injection of100, 200 and 500 mg, and tablets of 25 and 50 mg the skilled artisan isreferred to “Remington's Pharmaceutical Sciences” 15th Edition, chapter61, incorporate herein as a reference, for details on doses foradministration.

In practicing the present invention, one skilled in the art would knowthat superantigen therapy could be sequentially administered incombination with cyclophosphamide, for example, by first treating withcyclophosphamide. Once the effective cytostatic concentration ofcyclophosphamide has dropped in the patient below a functionalinhibitory level, the superantigen can be administered to the patient.

d) Melphalan

Melphalan, also known as alkeran, L-phenylalanine mustard, phenylalaninemustard, L-PAM, or L-sarcolysin, is a phenylalanine derivative ofnitrogen mustard. Melphalan is a bifunctional alkylating agent which isactive against selective human neoplastic diseases. It is knownchemically as 4-[bis(2-chloroethyl)amino]-L-phenylalanine.

Melphalan is the active L-isomer of the compound and was firstsynthesized in 1953 by Bergel and Stock; the D-isomer, known asmedphalan, is less active against certain animal tumors, and the doseneeded to produce effects on chromosomes is larger than that requiredwith the L-isomer. The racemic (DL-) form is known as merphalan orsarcolysin. Melphalan is insoluble in water and has a pKa1 of ˜2.1.Melphalan is available in tablet form for oral administration and hasbeen used to treat multiple myeloma.

Available evidence suggests that about one third to one half of thepatients with multiple myeloma show a favorable response to oraladministration of the drug.

Melphalan has been used in the treatment of epithelial ovariancarcinoma. One commonly employed regimen for the treatment of ovariancarcinoma has been to administer melphalan at a dose of 0.2 mg/kg dailyfor five days as a single course. Courses are repeated every four tofive weeks depending upon hematologic tolerance (Smith and Rutledge,1975; Young et al., 1978). Alternatively the dose of melphalan usedcould be as low as 0.05 mg/kg/day or as high as 3 mg/kg/day or any dosein between these doses or above these doses. Some variation in dosagewill necessarily occur depending on the condition of the subject beingtreated. The person responsible for administration will, in any event,determine the appropriate dose for the individual subject.

Absorption of melphalan from the gastrointestinal tract is variable; themean bioavailability is reported to be 56% but it may range from 25 to89%. Absorption is reduced by the presence of food. Following absorptionit is rapidly distributed throughout body water with a volume ofdistribution of about 0.5 liters per kg body-weight, and has beenreported to be inactivated mainly by spontaneous hydrolysis. Theterminal plasma half-life of melphalan has been reported to be of theorder of 40 to 140 minutes. Melphalan is excreted in the urine, about10% as unchanged drug. About 50 to 60% of an absorbed dose has beenstated to be protein bound initially, increasing to 80 to 90% after 12hours.

In practicing the present invention, one skilled in the art would knowthat superantigen therapy could be sequentially administered incombination with melphalan, for example, by first treating withmelphalan. Once the effective cytostatic concentration of melphalan hasdropped in the patient below a functional inhibitory level, thesuperantigen can be administered to the patient.

e) Carmustine

Carmustine (sterile carmustine) is one of the nitrosoureas used in thetreatment of certain neoplastic diseases. It is1,3bis(2-chloroethyl)-1-nitrosourea. It is lyophilized pale yellowflakes or congealed mass with a molecular weight of 214.06. It is highlysoluble in alcohol and lipids, and poorly soluble in water. Carmustineis administered by intravenous infusion after reconstitution asrecommended. Sterile carmustine is commonly available in 100 mg singledose vials of lyophilized material.

Although it is generally agreed that carmustine alkylates DNA and RNA,it is not cross resistant with other alkylators. As with othernitrosoureas, it may also inhibit several key enzymatic processes bycarbamoylation of amino acids in proteins.

Carmustine is indicated as palliative therapy as a single agent or inestablished combination therapy with other approved chemotherapeuticagents in brain tumors such as glioblastoma, brainstem glioma,medullobladyoma, astrocytoma, ependymoma, and metastatic brain tumors.Also it has been used in combination with prednisolone to treat multiplemyeloma. Carmustine has proved useful, in the treatment of Hodgkin'sDisease and in non-Hodgkin's lymphomas, as secondary therapy incombination with other approved drugs in patients who relapse whilebeing treated with primary therapy, or who fail to respond to primarytherapy.

The recommended dose of carmustine as a single agent in previouslyuntreated patients is 150 to 200 mg/m² intravenously every 6 weeks. Thismay be given as a single dose or divided into daily injections such as75 to 100 mg/m² on 2 successive days. The average terminal half-life isabout 22 minutes.

When carmustine is used in combination with other myelosuppressive drugsor in patients in whom bone marrow reserve is depleted, the doses shouldbe adjusted accordingly. Doses subsequent to the initial dose should beadjusted according to the hematologic response of the patient to thepreceding dose. It is of course understood that other doses may be usedin the present invention for example 10 mg/m², 20 mg/m², 30 mg/m² 40mg/m² 50 mg/m² 60 mg/m² 70 mg/m² 80 mg/m² 90 mg/m² 100 mg/m². Theskilled artisan is directed to, “Remington's Pharmaceutical Sciences”15th Edition, chapter 61. Some variation in dosage will necessarilyoccur depending on the condition of the subject being treated. Theperson responsible for administration will, in any event, determine theappropriate dose for the individual subject.

In practicing the present invention, one skilled in the art would knowthat superantigen therapy could be sequentially administered incombination with carmustine, for example, by first treating withcarmustine. Once the effective cytostatic concentration of carmustinehas dropped in the patient below a functional inhibitory level, thesuperantigen can be administered to the patient.

f) Lomustine

Lomustine is one of the nitrosoureas used in the treatment of certainneoplastic diseases. It is 1-(2-chloro-ethyl)-3-cyclohexyl-1nitrosourea. It is a yellow powder with the empirical formula ofC₉H₁₆ClN₃O₂ and a molecular weight of 233.71. Lomustine is soluble in10% ethanol (0.05 mg per mL) and in absolute alcohol (70 mg per mL).Lomustine is relatively insoluble in water (<0.05 mg per mL). It isrelatively unionized at a physiological pH. Inactive ingredients inlomustine capsules are: magnesium stearate and mannitol.

Although it is generally agreed that lomustine alkylates DNA and RNA, itis not cross resistant with other alkylators. As with othernitrosoureas, it may also inhibit several key enzymatic processes bycarbamoylation of amino acids in proteins.

Lomustine may be given orally. Following oral administration ofradioactive lomustine at doses ranging from 30 mg/m² to 100 mg/m², abouthalf of the radioactivity given was excreted in the form of degradationproducts within 24 hours.

The serum half-life of the metabolites ranges from 16 hours to 2 days.Tissue levels are comparable to plasma levels at 15 minutes afterintravenous administration.

Lomustine has been shown to be useful as a single agent in addition toother treatment modalities, or in established combination therapy withother approved chemotherapeutic agents in both primary and metastaticbrain tumors, in patients who have already received appropriate surgicaland/or radiotherapeutic procedures. It has also proved effective insecondary therapy against Hodgkin's Disease in combination with otherapproved drugs in patients who relapse while being treated with primarytherapy, or who fail to respond to primary therapy.

The recommended dose of lomustine in adults and children as a singleagent in previously untreated patients is 130 mg/m² as a single oraldose every 6 weeks. In individuals with compromised bone marrowfunction, the dose should be reduced to 100 mg/m² every 6 weeks. Whenlomustine is used in combination with other myelosuppressive drugs, thedoses should be adjusted accordingly. It is understood that other dosesmay be used for example, 20 mg/m² 30 mg/m², 40 mg/m², 50 mg/m², 60mg/m², 70 mg/m², 80 mg/m², 90 mg/m², 100 mg/m², 120 mg/m² or any dosesbetween these figures as determined by the clinician to be necessary forthe individual being treated.

In practicing the present invention, one skilled in the art would knowthat superantigen therapy could be sequentially administered incombination with lomustine, for example, by first treating withomustine. Once the effective cytostatic concentration of lomustine hasdropped in the patient below a functional inhibitory level, thesuperantigen can be administered to the patient.

3. Antimetabolites

Antimetabolites disrupt DNA and RNA synthesis. Unlike alkylating agents,antimetabolites specifically influence the cell cycle during S phase.Antimetabolites that are structural analogues of nucleotides areincorporated into cell components as if they were the essentialpyrimidine or purine, and as a consequence, disrupt the synthesis ofnucleic acids. Other antimetabolites disrupt essential enzymaticprocesses of metabolism. An example is the folate antagonist,5-fluorouracil, which disrupts vital folic acid metabolism. Exemplaryantimetabolites include, Cladribine, Cytarabine (Cytosine Arabinoside),Floxuridine (FUDR, 5-Fluorodeoxyuridine), Fludarabine, 5-Fluorouracil(5FU), Gemcitabine, Hydroxyurea, 6-Mercaptopurine (6 MP), Methotrexate(Amethopterin), 6-Thioguanine, Pentostatin, Pibobroman, Tegafur,Trimetrexate, Glucuronate.

Antimetabolites have used to combat chronic leukemias in addition totumors of breast, ovary and the gastrointestinal tract. Superantigenscan be used in combination with one or more of the antimetabolitesdescribed herein below.

a) 5-Fluorouracil and Related Compounds

5-Fluorouracil (5-FU) is the chemical name of5-fluoro-2,4(1H,3H)-pyrimidinedione. Its mechanism of action is thoughtto be by blocking the methylation reaction of deoxyuridylic acid tothymidylic acid. Thus, 5-FU interferes with the synthesis ofdeoxyribonucleic acid (DNA) and to a lesser extent inhibits theformation of ribonucleic acid (RNA). Since DNA and RNA are essential forcell division and proliferation, it is thought that the effect of 5-FUis to create a thymidine deficiency leading to cell death. Thus, theeffect of 5-FU is found in cells that rapidly divide, a characteristicof metastatic cancers.

The elimination half-life of 5-FU is 6 to 20 minutes and isdose-dependent. The metabolism of 5-FU occurs mainly in the liver andresults in degradation products (e.g., carbon dioxide, urea,a-fluoro-b-alanine) which are inactive. Approximately 15% of the dose isexcreted intact in the urine in 6 hours and over 90% of this is excretedin the first hour; 60 to 80% is excreted as respiratory carbon dioxidein 8 to 12 hours.

In practicing the present invention, one skilled in the art would knowthat superantigen therapy could be sequentially administered incombination with 5-FU, for example, by first treating with 5-FU. Oncethe effective cytostatic concentration of 5-FU has dropped in thepatient below a functional inhibitory level, the superantigen can beadministered to the patient.

Other compounds that are related to 5-FU are also included within thepresent invention (e.g., but not limited to capecitabine XELODA®(Roche)).

b) Gemcitabine

Gemcitabine is commonly used to for non-small cell lung carcinomas andpancreatic carcinoma. Gemcitabine dosages of 1000 or 1250 mg/m² areadministered by 30-minute IV infusion once weekly for 3 weeks followedby 1 week of rest. Various dosage schedules have been studied for thecombination of gemcitabine with cisplatin for the treatment of advancednon-small cell lung cancer; gemcitabine dosages of 1000 mg/m²administered once weekly for 3 weeks on a 4-week cycle or 1250 mg/m²administered once weekly for 2 weeks on a 3-week cycle have been used inlarge randomized trials. Other dosage schedules for gemcitabine (e.g.,higher doses, lower doses administered over longer infusion periods) canalso be used. For example, doses of 65 mg/kg may also be administered.

The half-life of gemcitabine depends upon the length of the infusionperiod, age and gender. For example, gemcitabine half-life for shortinfusions range from 32 to 94 minutes, and the value for long infusionsvary from 245 to 638 minutes. The lower clearance in women and theelderly results in higher concentrations of gemcitabine for any givendose. In practicing the present invention, one skilled in the art wouldknow that superantigen therapy could be sequentially administered incombination with gemcitabine, for example, by first treating withgemcitabine. Once the effective cytostatic concentration of gemcitabinehas dropped in the patient below a functional inhibitory level, thesuperantigen can be administered to the patient.

c) Pemetrexed

Pemetrexed (Alimta®) is an antifolate agent. More specifically,pemetrexed and its polyglutamates inhibit at least four enzymes involvedin folate metabolism and DNA synthesis; this multitargeted action isspeculated to limit development of drug resistance. It is approved forthe treatment of patients with malignant pleural mesothelioma who arenot candidates for surgical resection. The drug has also shownanti-tumor activity in non-small-cell lung cancer, colorectal carcinoma,breast cancer, and several other malignancies.

Pemetrexed is administered intravenously (IV) at a dose of 500milligrams/square meter (mg/m²) over 10 minutes on day 1 of each 21-daycycle. After intravenous doses (ranging from 0.2 to 838 mg/m²),pemetrexed has a volume of distribution of 16.1 liters, suggestinglimited distribution in tissues, and a clearance of 91.8milliliters/minute. Protein binding is about 81%. Metabolism appearsminimal, and most of a dose is excreted unchanged in the urine. Thehalf-life is about 3.5 hours.

In practicing the present invention, one skilled in the art would knowthat superantigen therapy could be sequentially administered incombination with pemetrexed, for example, by first treating withpemetrexed. Once the effective cytostatic concentration of pemetrexedhas dropped in the patient below a functional inhibitory level, thesuperantigen can be administered to the patient.

4. Anti-Tumor Antibiotics

Anti-tumor antibiotics have both antimicrobial and cytotoxic activity.These drugs also interfere with DNA by chemically inhibiting enzymes andmitosis or altering cellular membranes. These agents are not phasespecific so they work in all phases of the cell cycle. Thus, they arewidely used for a variety of cancers. Most anti-tumor antibioticsintercalate between DNA base pairs and disturb the synthesis and/orfunction of nucleic acids. However, a different mechanism is ascribed tobleomycin. Bleomycin apparently binds to DNA and results insingle-strand breaks and double-strand scissions, thereby disrupting DNAsynthesis. Doxorubicin not only intercalates between base pairs, butalso alkylates macromolecules. Daunorubicin, doxorubicin, and theirderivatives, belong to a subclass of anti-tumor antibiotics calledanthracyclines. Exemplary anti-tumor antibiotics include, Aclarubicin,Bleomycin, Dactinomycin (Actinomycin D), Daunorubicin, Doxorubicin(Adriamycin), Epirubicin, Idarubicin, Mitomycin C, Mitoxantrone,Plicamycin (Mithramycin). Superantigens can be used in combination withone or more of the anti-tumor antibotics described herein below.

a) Doxorubicin

Doxorubicin hydrochloride, 5,12-Naphthacenedione, (8s-cis)-10-[(3-amino-2,3,6-trideoxy-a-L-lyxo-hexopyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-hydrochloride(hydroxydaunorubicin hydrochloride, Adriamycin) is used in a wideantineoplastic spectrum. It binds to DNA and inhibits nucleic acidsynthesis, inhibits mitosis and promotes chromosomal aberrations.

Administered alone, it is the drug of first choice for the treatment ofthyroid adenoma and primary hepatocellular carcinoma. It is a componentof 31 first-choice combinations for the treatment of ovarian,endometrial and breast tumors, bronchogenic oat-cell carcinoma,non-small cell lung carcinoma, gastric adenocarcinoma, retinoblastoma,neuroblastoma, mycosis fungoides, pancreatic carcinoma, prostaticcarcinoma, bladder carcinoma, myeloma, diffuse histiocytic lymphoma,Wilms' tumor, Hodgkin's disease, adrenal tumors, osteogenic sarcoma softtissue sarcoma, Ewing's sarcoma, rhabdomyosarcoma and acute lymphocyticleukemia. It is an alternative drug for the treatment of islet cell,cervical, testicular and adrenocortical cancers. It is also animmunosuppressant.

Doxorubicin is absorbed poorly and must be administered intravenously.The pharmacokinetics are multicompartmental. Distribution phases havehalf-lives of 12 minutes and 3.3 hr. The half-life is about 30 hr. Fortyto 50% is secreted into the bile. Most of the remainder is metabolizedin the liver, partly to an active metabolite (doxorubicinol), but a fewpercent is excreted into the urine. In the presence of liver impairment,the dose should be reduced.

Appropriate doses are, intravenous, adult, 60 to 75 mg/m² at 21-dayintervals or 25 to 30 mg/m² on each of 2 or 3 successive days repeatedat 3- or 4-wk intervals or 20 mg/m² once a week. The lowest dose shouldbe used in elderly patients, when there is prior bone-marrow depressioncaused by prior chemotherapy or neoplastic marrow invasion, or when thedrug is combined with other myelopoietic suppressant drugs. The doseshould be reduced by 50% if the serum bilirubin lies between 1.2 and 3mg/dL and by 75% if above 3 mg/dL. The lifetime total dose should notexceed 550 mg/m2 in patients with normal heart function and 400 mg/m² inpersons having received mediastinal irradiation. Alternatively, 30 mg/m²on each of 3 consecutive days, repeated every 4 wk. Exemplary doses maybe 10 mg/m², 20 mg/m², 30 mg/m², 50 mg/m², 100 mg/m², 150 mg/m², 175mg/m², 200 mg/m², 225 mg/m², 250 mg/m², 275 mg/m², 300 mg/m², 350 mg/m²,400 mg/m², 425 mg/m², 450 mg/m², 475 mg/m², 500 mg/m². Of course, all ofthese dosages are exemplary, and any dosage in-between these points isalso expected to be of use in the invention.

In practicing the present invention, one skilled in the art would knowthat superantigen therapy could be sequentially administered incombination with doxorubicin, for example, by first treating withdoxorubicin. Once the effective cytostatic concentration of doxorubicinhas dropped in the patient below a functional inhibitory level, thesuperantigen can be administered to the patient.

b) Daunorubicin

Daunorubicin hydrochloride, 5,12-Naphthacenedione,(8S-cis)-8-acetyl-10-[(3-amino-2,3,6-trideoxy-a-L-lyxo-hexanopyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-10-methoxy-,hydrochloride; also termed cerubidine and available from Wyeth.Daunorubicin intercalates into DNA, blocks DAN-directed RNA polymeraseand inhibits DNA synthesis. It can prevent cell division in doses thatdo not interfere with nucleic acid synthesis.

In combination with other drugs it is included in the first-choicechemotherapy of acute myelocytic leukemia in adults (for induction ofremission), acute lymphocytic leukemia and the acute phase of chronicmyelocytic leukemia. Oral absorption is poor, and it must be givenintravenously. The half-life of distribution is 45 minutes and ofelimination, about 19 hr. The half-life of its active metabolite,daunorubicinol, is about 27 hr. Daunorubicin is metabolized mostly inthe liver and also secreted into the bile (ca 40%). Dosage must bereduced in liver or renal insufficiencies.

Suitable doses are (base equivalent), intravenous adult, younger than 60yr. 45 mg/m²/day (30 mg/m² for patients older than 60 yr.) for 1, 2 or 3days every 3 or 4 wk or 0.8 mg/kg/day for 3 to 6 days every 3 or 4 wk;no more than 550 mg/m2 should be given in a lifetime, except only 450mg/m2 if there has been chest irradiation; children, 25 mg/m² once aweek unless the age is less than 2 yr. or the body surface less than 0.5m, in which case the weight-based adult schedule is used. It isavailable in injectable dosage forms (base equivalent) 20 mg (as thebase equivalent to 21.4 mg of the hydrochloride). Exemplary doses may be10 mg/m², 20 mg/m², 30 mg/m², 50 mg/m², 100 mg/m², 150 mg/m², 175 mg/m²,200 mg/m², 225 mg/m², 250 mg/m², 275 mg/m², 300 mg/m², 350 mg/m², 400mg/m², 425 mg/m², 450 mg/m², 475 mg/m², 500 mg/m². Of course, all ofthese dosages are exemplary, and any dosage in-between these points isalso expected to be of use in the invention.

In practicing the present invention, one skilled in the art would knowthat superantigen therapy could be sequentially administered incombination with daunorubicin, for example, by first treating withdaunorubicin. Once the effective cytostatic concentration ofdaunorubicin has dropped in the patient below a functional inhibitorylevel, the superantigen can be administered to the patient.

c) Mitomycin

Mitomycin (also known as mutamycin and/or mitomycin-C) is an antibioticisolated from the broth of Streptomyces caespitosus which has been shownto have anti-tumor activity. The compound is heat stable, has a highmelting point, and is freely soluble in organic solvents.

Mitomycin selectively inhibits the synthesis of deoxyribonucleic acid(DNA). The guanine and cytosine content correlates with the degree ofmitomycin-induced cross-linking. At high concentrations of the drug,cellular RNA and protein synthesis are also suppressed.

In humans, mitomycin is rapidly cleared from the serum after intravenousadministration. Time required to reduce the serum concentration by 50%after a 30 mg bolus injection is 17 minutes. After injection of 30 mg,20 mg, or 10 mg I.V., the maximal serum concentrations were 2.4 mg/mL,1.7 mg/mL, and 0.52 mg/mL, respectively. Clearance is effected primarilyby metabolism in the liver, but metabolism occurs in other tissues aswell. The rate of clearance is inversely proportional to the maximalserum concentration because, it is thought, of saturation of thedegradative pathways. Approximately 10% of a dose of mitomycin isexcreted unchanged in the urine. Since metabolic pathways are saturatedat relatively low doses, the percent of a dose excreted in urineincreases with increasing dose. In children, excretion of intravenouslyadministered mitomycin is similar.

In practicing the present invention, one skilled in the art would knowthat superantigen therapy could be sequentially administered incombination with mitomycin, for example, by first treating withmitomycin. Once the effective cytostatic concentration of mitomycin hasdropped in the patient below a functional inhibitory level, thesuperantigen can be administered to the patient.

d) Actinomycin D

Actinomycin D (Dactinomycin) [50-76-0]; C₆₂H₈₆N₁₂O₁₆ (1255.43) is anantineoplastic drug that inhibits DNA-dependent RNA polymerase. It is acomponent of first-choice combinations for treatment of choriocarcinoma,embryonal rhabdomyosarcoma, testicular tumor and Wilms' tumor. Tumorsthat fail to respond to systemic treatment sometimes respond to localperfusion. Dactinomycin potentiates radiotherapy. It is a secondary(efferent) immunosuppressive.

Actinomycin D is used in combination with primary surgery, radiotherapy,and other drugs, particularly vincristine and cyclophosphamide.Antineoplastic activity has also been noted in Ewing's tumor, Kaposi'ssarcoma, and soft-tissue sarcomas. Dactinomycin can be effective inwomen with advanced cases of choriocarcinoma. It also producesconsistent responses in combination with chlorambucil and methotrexatein patients with metastatic testicular carcinomas. A response maysometimes be observed in patients with Hodgkin's disease andnon-Hodgkin's lymphomas. Dactinomycin has also been used to inhibitimmunological responses, particularly the rejection of renaltransplants.

Half of the dose is excreted intact into the bile and 10% into theurine; the half-life is about 36 hr. The drug does not pass theblood-brain barrier. Actinomycin D is supplied as a lyophilized powder(0.5 mg in each vial). The usual daily dose is 10 to 15 mg/kg; this isgiven intravenously for 5 days; if no manifestations of toxicity areencountered, additional courses may be given at intervals of 3 to 4weeks. Daily injections of 100 to 400 mg have been given to children for10 to 14 days; in other regimens, 3 to 6 mg/kg, for a total of 125mg/kg, and weekly maintenance doses of 7.5 mg/kg have been used.Although it is safer to administer the drug into the tubing of anintravenous infusion, direct intravenous injections have been given,with the precaution of discarding the needle used to withdraw the drugfrom the vial in order to avoid subcutaneous reaction. Exemplary dosesmay be 100 mg/m², 150 mg/m², 175 mg/m², 200 mg/m², 225 mg/m², 250 mg/m²,275 mg/m², 300 mg/m², 350 mg/m², 400 mg/m², 425 mg/m², 450 mg/m², 475mg/m², 500 mg/m². Of course, all of these dosages are exemplary, and anydosage in-between these points is also expected to be of use in theinvention.

In practicing the present invention, one skilled in the art would knowthat superantigen therapy could be sequentially administered incombination with actinomycin D, for example, by first treating withactinomycin D. Once the effective cytostatic concentration ofactinomycin D has dropped in the patient below a functional inhibitorylevel, the superantigen can be administered to the patient.

e) Bleomycin

Bleomycin is a mixture of cytotoxic glycopeptide antibiotics isolatedfrom a strain of Streptomyces verticillus. Although the exact mechanismof action of bleomycin is unknown, available evidence would seem toindicate that the main mode of action is the inhibition of DNA synthesiswith some evidence of lesser inhibition of RNA and protein synthesis.

In mice, high concentrations of bleomycin are found in the skin, lungs,kidneys, peritoneum, and lymphatics. Tumor cells of the skin and lungshave been found to have high concentrations of bleomycin in contrast tothe low concentrations found in hematopoietic tissue. The lowconcentrations of bleomycin found in bone marrow may be related to highlevels of bleomycin degradative enzymes found in that tissue.

In patients with a creatinine clearance of >35 mL per minute, the serumor plasma terminal elimination half-life of bleomycin is approximately115 minutes. In patients with a creatinine clearance of <35 mL perminute, the plasma or serum terminal elimination half-life increasesexponentially as the creatinine clearance decreases. In humans, 60% to70% of an administered dose is recovered in the urine as activebleomycin. Bleomycin may be given by the intramuscular, intravenous, orsubcutaneous routes. It is freely soluble in water.

Bleomycin should be considered a palliative treatment. It has been shownto be useful in the management of the following neoplasms either as asingle agent or in proven combinations with other approvedchemotherapeutic agents in squamous cell carcinoma such as head and neck(including mouth, tongue, tonsil, nasopharynx, oropharynx, sinus,palate, lip, buccal mucosa, gingiva, epiglottis, larynx), skin, penis,cervix, and vulva. It has also been used in the treatment of lymphomasand testicular carcinoma.

Because of the possibility of an anaphylactoid reaction, lymphomapatients should be treated with two units or less for the first twodoses. If no acute reaction occurs, then the regular dosage schedule maybe followed.

Improvement of Hodgkin's Disease and testicular tumors is prompt andnoted within 2 weeks. If no improvement is seen by this time,improvement is unlikely. Squamous cell cancers respond more slowly,sometimes requiring as long as 3 weeks before any improvement is noted.

In practicing the present invention, one skilled in the art would knowthat superantigen therapy could be sequentially administered incombination with bleomycin, for example, by first treating withbleomycin. Once the effective cytostatic concentration of bleomycin hasdropped in the patient below a functional inhibitory level, thesuperantigen can be administered to the patient.

5. Mitotic Inhibitors

Mitotic inhibitors include plant alkaloids and other natural agents thatcan inhibit either protein synthesis required for cell division ormitosis. They operate during a specific phase during the cell cycle.Mitotic inhibitors include among others the vinca alkaloids (vincristineand vinblastine) which are mitotic spindle inhibitors and theepipodophyllotoxins (teniposide and etoposide) which are DNAtopoisomerase II inhibitors. Mitotic spindle inhibitors bind tomicrotubular proteins and block their ability to polymerize ordepolymerize, a process which halts nuclear division. DNA topoisomeraseII inhibitors block religation of double strand DNA breaks (e.g., sisterchromatid separation or cleaved DNA). Examples include Etoposide (VP-16,VePesid), Paclitaxel (TAXOL®), Docetaxel (Taxotere) Teniposide (VM-26,Vumon), Vinblastine, Vincristine, Vindesine, Topotecan, and Irinotecan.Superantigens can be used in combination with one or more of the mitoticinhibitors described herein below.

a) Paclitaxel

Paclitaxel (TAXOL®) is an experimental antimitotic agent, isolated fromthe bark of the ash tree, Taxus brevifolia. It binds to tubulin (at asite distinct from that used by the vinca alkaloids) and promotes theassembly of microtubules. Paclitaxel is known to have activity againstmalignant melanoma and carcinoma of the ovary. Maximal doses are 30mg/m² per day for 5 days or 210 to 250 mg/m² given once every 3 weeks.Of course, all of these dosages are exemplary, and any dosage in-betweenthese points is also expected to be of use in the invention.

In patients treated with doses of 135 and 175 mg/m² given as 3 and 24hour infusions, half-life ranges from 3.0 to 52.7 hours, and total bodyclearance ranges from 11.6 to 24.0 L/h/m². Mean steady state volume ofdistribution following single dose infusion of 135 and 175 mg/m² rangesfrom 198 to 688 L/m², indicating extensive extravascular distributionand/or tissue binding. The volume of distribution is reduced in femalesubjects. Following 3 hour infusions of 175 mg/m², mean terminalhalf-life is estimated to be 9.9 hours; mean total body clearance is12.4 L/h/m².

In practicing the present invention, one skilled in the art would knowthat superantigen therapy could be sequentially administered incombination with paclitaxel, for example, by first treating withpaclitaxel. Once the effective cytostatic concentration of paclitaxelhas dropped in the patient below a functional inhibitory level, thesuperantigen can be administered to the patient.

b) Docetaxel

Docetaxel (TAXOTERE®) is given as a treatment for some types of cancer.

It is most commonly used to treat breast cancer and non-small cell lungcancer, but may be used for other types of cancer. Docetaxel isadministered by IV infusion over a 1-hour period under ambient roomtemperature and lighting conditions. The dosage of docetaxel ranges from20 mg/m² to 115 mg/m². The half-life is about 13.5+/−7.5 hours.

In practicing the present invention, one skilled in the art would knowthat superantigen therapy could be sequentially administered incombination with docetaxel, for example, by first treating withdocetaxel. Once the effective cytostatic concentration of docetaxel hasdropped in the patient below a functional inhibitory level, thesuperantigen can be administered to the patient.

c) Vinblastine

Vinblastine is another example of a plant alkyloid that can be used incombination with an superantigen for the treatment of cancer andprecancer. When cells are incubated with vinblastine, dissolution of themicrotubules occurs.

Unpredictable absorption has been reported after oral administration ofvinblastine or vincristine. At the usual clinical doses the peakconcentration of each drug in plasma is approximately 0.4 mM.Vinblastine and vincristine bind to plasma proteins. They areextensively concentrated in platelets and to a lesser extent inleukocytes and erythrocytes.

After intravenous injection, vinblastine has a multiphasic pattern ofclearance from the plasma; after distribution, drug disappears fromplasma with half-lives of approximately 1 and 20 hours. Vinblastine ismetabolized in the liver to biologically activate derivativedesacetylvinblastine. Approximately 15% of an administered dose isdetected intact in the urine, and about 10% is recovered in the fecesafter biliary excretion. Doses should be reduced in patients withhepatic dysfunction. At least a 50% reduction in dosage is indicated ifthe concentration of bilirubin in plasma is greater than 3 mg/dl (about50 mM).

Vinblastine sulfate is available in preparations for injection. The drugis given intravenously; special precautions must be taken againstsubcutaneous extravasation, since this may cause painful irritation andulceration. The drug should not be injected into an extremity withimpaired circulation. After a single dose of 0.3 mg/kg of body weight,myelosuppression reaches its maximum in 7 to 10 days. If a moderatelevel of leukopenia (approximately 3000 cells/mm³) is not attained, theweekly dose may be increased gradually by increments of 0.05 mg/kg ofbody weight. In regimens designed to cure testicular cancer, vinblastineis used in doses of 0.3 mg/kg every 3 weeks irrespective of blood cellcounts or toxicity.

The most important clinical use of vinblastine is with bleomycin andcisplatin in the curative therapy of metastatic testicular tumors.Beneficial responses have been reported in various lymphomas,particularly Hodgkin's disease, where significant improvement may benoted in 50 to 90% of cases. The effectiveness of vinblastine in a highproportion of lymphomas is not diminished when the disease is refractoryto alkylating agents. It is also active in Kaposi's sarcoma,neuroblastoma, and Letterer-Siwe disease (histiocytosis X), as well asin carcinoma of the breast and choriocarcinoma in women.

Doses of vinblastine will be determined by the clinician according tothe individual patients need. 0.1 to 0.3 mg/kg can be administered or1.5 to 2 mg/m² can also be administered. Alternatively, 0.1 mg/m², 0.12mg/m², 0.14 mg/m², 0.15 mg/m², 0.2 mg/m², 0.25 mg/m², 0.5 mg/m², 1.0mg/m², 1.2 mg/m², 1.4 mg/m², 1.5 mg/m², 2.0 mg/m², 2.5 mg/m², 5.0 mg/m²,6 mg/m², 8 mg/m², 9 mg/m², 10 mg/m², 20 mg/m², can be given. Of course,all of these dosages are exemplary, and any dosage in-between thesepoints is also expected to be of use in the invention.

In practicing the present invention, one skilled in the art would knowthat superantigen therapy could be sequentially administered incombination with vinblastine, for example, by first treating withvinblastine. Once the effective cytostatic concentration of vinblastinehas dropped in the patient below a functional inhibitory level, thesuperantigen can be administered to the patient.

d) Vincristine

Vincristine blocks mitosis and produces metaphase arrest. It seemslikely that most of the biological activities of this drug can beexplained by its ability to bind specifically to tubulin and to blockthe ability of protein to polymerize into microtubules. Throughdisruption of the microtubules of the mitotic apparatus, cell divisionis arrested in metaphase. The inability to segregate chromosomescorrectly during mitosis presumably leads to cell death.

The relatively low toxicity of vincristine for normal marrow cells andepithelial cells make this agent unusual among anti-neoplastic drugs,and it is often included in combination with other myelosuppressiveagents.

Unpredictable absorption has been reported after oral administration ofvinblastine or vincristine. At the usual clinical doses the peakconcentration of each drug in plasma is approximately 0.4 mM.

Vinblastine and vincristine bind to plasma proteins. They areextensively concentrated in platelets and to a lesser extent inleukocytes and erythrocytes.

Vincristine has a multiphasic pattern of clearance from the plasma; theterminal half-life is about 24 hours. The drug is metabolized in theliver, but no biologically active derivatives have been identified.Doses should be reduced in patients with hepatic dysfunction. At least a50% reduction in dosage is indicated if the concentration of bilirubinin plasma is greater than 3 mg/dl (about 50 mM).

Vincristine sulfate is available as a solution (1 mg/ml) for intravenousinjection. Vincristine used together with corticosteroids is presentlythe treatment of choice to induce remissions in childhood leukemia; theoptimal dosages for these drugs appear to be vincristine, intravenously,2 mg/m² of body-surface area, weekly, and prednisone, orally, 40 mg/m²,daily. Adult patients with Hodgkin's disease or non-Hodgkin's lymphomasusually receive vincristine as a part of a complex protocol. When usedin the MOPP regimen, the recommended dose of vincristine is 1.4 mg/m².High doses of vincristine seem to be tolerated better by children withleukemia than by adults, who may experience sever neurological toxicity.Administration of the drug more frequently than every 7 days or athigher doses seems to increase the toxic manifestations withoutproportional improvement in the response rate. Precautions should alsobe used to avoid extravasation during intravenous administration ofvincristine. Vincristine (and vinblastine) can be infused into thearterial blood supply of tumors in doses several times larger than thosethat can be administered intravenously with comparable toxicity.

Vincristine has been effective in Hodgkin's disease and other lymphomas.Although it appears to be somewhat less beneficial than vinblastine whenused alone in Hodgkin's disease, when used with mechlorethamine,prednisone, and procarbazine (the so-called MOPP regimen), it is thepreferred treatment for the advanced stages (III and IV) of thisdisease. In non-Hodgkin's lymphomas, vincristine is an important agent,particularly when used with cyclophosphamide, bleomycin, doxorubicin,and prednisone. Vincristine is more useful than vinblastine inlymphocytic leukemia. Beneficial response have been reported in patientswith a variety of other neoplasms, particularly Wilms' tumor,neuroblastoma, brain tumors, rhabdomyosarcoma, and carcinomas of thebreast, bladder, and the male and female reproductive systems.

Doses of vincristine for use will be determined by the clinicianaccording to the individual patients need. 0.01 to 0.03 mg/kg or 0.4 to1.4 mg/m² can be administered or 1.5 to 2 mg/m² can also beadministered. Alternatively 0.02 mg/m2, 0.05 mg/m2, 0.06 mg/m2, 0.07mg/m², 0.08 mg/m², 0.1 mg/m², 0.12 mg/m², 0.14 mg/m², 0.15 mg/m², 0.2mg/m², 0.25 mg/m² can be given as a constant intravenous infusion. Ofcourse, all of these dosages are exemplary, and any dosage in-betweenthese points is also expected to be of use in the invention.

In practicing the present invention, one skilled in the art would knowthat superantigen therapy could be sequentially administered incombination with vincristine, for example, by first treating withvincristine. Once the effective cytostatic concentration of vincristinehas dropped in the patient below a functional inhibitory level, thesuperantigen can be administered to the patient.

e) Etoposide (VP16)

VP 16 is also known as etoposide and is used primarily for treatment oftesticular tumors, in combination with bleomycin and cisplatin, and incombination with cisplatin for small-cell carcinoma of the lung. It isalso active against non-Hodgkin's lymphomas, acute nonlymphocyticleukemia, carcinoma of the breast, and Kaposi's sarcoma associated withacquired immunodeficiency syndrome (AIDS).

VP16 is available as a solution (20 mg/ml) for intravenousadministration and as 50-mg, liquid-filled capsules for oral use. Forsmall-cell carcinoma of the lung, the intravenous dose (in combinationtherapy) is can be as much as 100 mg/m² or as little as 2 mg/m²,routinely 35 mg/m², daily for 4 days, to 50 mg/m², daily for 5 days havealso been used. When given orally, the dose should be doubled. Hence thedoses for small cell lung carcinoma may be as high as 200-250 mg/m². Theintravenous dose for testicular cancer (in combination therapy) is 50 to100 mg/m² daily for 5 days, or 100 mg/m² on alternate days, for threedoses. Cycles of therapy are usually repeated every 3 to 4 weeks. Thedrug should be administered slowly during a 30- to 60-minute infusion inorder to avoid hypotension and bronchospasm, which are probably due tothe solvents used in the formulation.

On intravenous administration, the disposition of VP16 is best describedas a biphasic process with a distribution half-life of about 1.5 hoursand terminal elimination half-life ranging from 4 to 11 hours. Totalbody clearance values range from 33 to 48 mL/min or 16 to 36 mL/min/m²and, like the terminal elimination half-life, are independent of doseover a range 100-600 mg/m².

In practicing the present invention, one skilled in the art would knowthat superantigen therapy could be sequentially administered incombination with VP 16, for example, by first treating with VP16. Oncethe effective cytostatic concentration of VP16 has dropped in thepatient below a functional inhibitory level, the superantigen can beadministered to the patient.

6. Platinum Based Compounds

Platinum-based compounds are among the most active chemotherapeuticagents available. They are effective against a multitude of cancers.Platinum-containing antineoplastic agents, such as carboplatin andcisplatin, appear to exert their effects by binding to DNA, therebyinhibiting DNA synthesis. The drugs are cycle-phase nonspecific.Carboplatin and cisplatin appear to act on tumor cells by the samegeneral molecular mechanisms and, once bound to DNA, have virtually thesame action. Although the principal mechanism of action of the drugsappears to be inhibition of DNA synthesis, other mechanisms also areinvolved in their antineoplastic activity. Superantigens can be used incombination with one or more of the platinum-based compounds describedherein below.

a) Cisplatin

Cisplatin has been widely used to treat cancers such as metastatictesticular or ovarian carcinoma, advanced bladder cancer, head or neckcancer, cervical cancer, lung cancer or other tumors. Cisplatin can beused alone or in combination with other agents, with efficacious dosesused in clinical applications of 15-20 mg/m² for 5 days every threeweeks for a total of three courses. Exemplary doses may be 0.50 mg/m²,1.0 mg/m², 1.50 mg/m², 1.75 mg/m², 2.0 mg/m², 3.0 mg/m², 4.0 mg/m², 5.0mg/m², 10 mg/m². Of course, all of these dosages are exemplary, and anydosage in-between these points is also expected to be of use in theinvention.

Cisplatin is not absorbed orally and must therefore be delivered viainjection intravenously, subcutaneously, intratumorally orintraperitoneally.

Plasma concentrations of cisplatin decay monoexponentially with ahalf-life of about 20 to 30 minutes following bolus administration of 50or 100 mg/m² doses. Monoexponential decay and plasma half-lives of about0.5 hour are also seen following two hour or seven hour infusions of 100mg/m².

Following cisplatin doses of 20 to 120 mg/m², the concentrations ofplatinum are highest in liver, prostate, and kidney, somewhat lower inbladder, muscle, testicle, pancreas, and spleen and lowest in bowel,adrenal, heart, lung, cerebrum, and cerebellum. Platinum is present intissues for as long as 180 days after the last administration. Maximumred blood cell concentrations of platinum are reached within 90 to 150minutes after a 100 mg/m² dose of cisplatin and decline in a biphasicmanner with a half-life of 36 to 47 days.

In practicing the present invention, one skilled in the art would knowthat superantigen therapy could be sequentially administered incombination with cisplatin, for example, by first treating withcisplatin. Once the effective cytostatic concentration of cisplatin hasdropped in the patient below a functional inhibitory level, thesuperantigen can be administered to the patient.

b) Carboplatin

Carboplatin and cisplatin are associated with different toxicityprofiles and carboplatin may be effective in patients withplatinum-responsive tumors who are unable to tolerate cisplatin becauseof renal impairment, refractory nausea, hearing impairment, orneuropathy. It has been suggested that while carboplatin may bepreferred in patients with renal failure or patients at high risk forototoxicity or neurotoxicity, cisplatin may be preferred in patients whohave decreased bone marrow reserve, a high risk of sepsis, or requireanticoagulation therapy.

Dosage of carboplatin is based on the clinical, renal, and hematologicresponse and tolerance of the patient in order to obtain optimumtherapeutic response with minimum adverse effects. While initialcarboplatin dosage are based on body surface area, dosage may be moreaccurately calculated using formula dosing methods based on thepatient's renal function.

Carboplatin decays in a biphasic manner after a 30-minute intravenousinfusion of 300 to 500 mg/m². The initial plasma half-life (alpha) wasfound to be 1.1 to 2 hours (N=6), and the postdistribution plasmahalf-life (beta) was found to be 2.6 to 5.9 hours (N=6). The total bodyclearance, apparent volume of distribution and mean residence time forcarboplatin are 4.4 L/hour, 16 L and 3.5 hours, respectively. Platinumfrom carboplatin becomes irreversibly bound to plasma proteins and isslowly eliminated with a minimum half-life of 5 days.

In practicing the present invention, one skilled in the art would knowthat superantigen therapy could be sequentially administered incombination with carboplatin, for example, by first treating withcarboplatin. Once the effective cytostatic concentration of carboplatinhas dropped in the patient below a functional inhibitory level, thesuperantigen can be administered to the patient.

c) Oxaliplatin

Oxaliplatin (Eloxatin®) organoplatinum complex in which the platinumatom is complexed with 1,2-diaminocyclohexane (DACH) and with an oxalateligand as a leaving group (Scheeff E D, Briggs J M, Howell S B.Molecular modeling of the intrastrand guanine-guanine DNA adductsproduced by cisplatin and oxaliplatin. Mol. Pharmacol. 1999;56:633-643).

Oxaliplatin is administered by infusion into a vein over at least 2hours by a health care professional. It is typically given as one dose(85 mg/m2) every 2 weeks, along with other drugs (e.g., 5-fluorouraciland leucovorin). This cycle is repeated every 2 weeks. The dose andfrequency is based on the blood count and response to previous doses ofthe subject. The terminal half life is about 273±19 h.

In practicing the present invention, one skilled in the art would knowthat superantigen therapy could be sequentially administered incombination with oxaliplatin, for example, by first treating withoxaliplatin. Once the effective cytostatic concentration of oxaliplatinhas dropped in the patient below a functional inhibitory level, thesuperantigen can be administered to the patient.

B. Radiotherapy

In further embodiments, it is envisioned that the superantigen therapyof the present invention can be used in combination with radiotherapy.X-rays, γ-rays, and/or the directed delivery of radioisotopes to tumorcells have been commonly used to treat hyperproliferative disease, morespecifically cancer. It is known that these factors cause DNA damage.Other forms of DNA damaging factors are also contemplated such asmicrowaves and UV-irradiation. It is most likely that all of thesefactors effect a broad range of damage on DNA, on the precursors of DNA,on the replication and repair of DNA, and on the assembly andmaintenance of chromosomes. Dosage ranges for X-rays range from dailydoses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk),to single doses of 2000 to 6000 roentgens. Dosage ranges forradioisotopes vary widely, and depend on the half-life of the isotope,the strength and type of radiation emitted, and the uptake by theneoplastic cells.

C. Surgery

Approximately 60% of persons with cancer will undergo surgery of sometype, which includes preventative, diagnostic or staging, curative andpalliative surgery. Curative surgery is a cancer treatment that may beused in conjunction with other therapies, such as the treatment of thepresent invention, chemotherapy, radiotherapy, hormonal therapy, genetherapy, immunotherapy and/or alternative therapies. Thus, in furtherembodiments, it is envisioned that the superantigen therapy of thepresent invention can be used in combination with surgery to treat acancer.

Curative surgery includes resection in which all or part of canceroustissue is physically removed, excised, and/or destroyed. Tumor resectionrefers to physical removal of at least part of a tumor. In addition totumor resection, treatment by surgery includes laser surgery,cryosurgery, electrosurgery, and miscopically controlled surgery (Mohs'surgery). It is further contemplated that the present invention may beused in conjunction with removal of superficial cancers, precancers, orincidental amounts of normal tissue.

Upon excision of part of all of cancerous cells, tissue, or tumor, acavity may be formed in the body. Treatment may be accomplished byperfusion, direct injection or local application of the area with anadditional anticancer therapy. Such treatment may be repeated, forexample, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. Thesetreatments may be of varying dosages as well.

D. Targeted Molecules Including Antibodies

In further embodiments, it is envisioned that the superantigen therapyof the present invention can be used in combination with targetedmolecules including, but not limited to, antibodies. For example, anantibody specific for some marker on the surface of a tumor cell. Forexample, the antibody alone may serve as an effector of therapy or itmay recruit other cells to actually effect cell killing. The antibodyalso may be conjugated to a drug or toxin (e.g., a chemotherapeutic, aradionuclide, a ricin A chain, a cholera toxin, a pertussis toxin, etc.)and serve merely as a targeting agent. Such antibody conjugates arecalled immunotoxins, and are well known in the art (see U.S. Pat. No.5,686,072, U.S. Pat. No. 5,578,706, U.S. Pat. No. 4,792,447, U.S. Pat.No. 5,045,451, U.S. Pat. No. 4,664,911, and U.S. Pat. No. 5,767,072,each incorporated herein by reference).

Preferably, human monoclonal antibodies are employed in passiveimmunotherapy, as they produce few or no side effects in the patient.For example, such antibodies have been developed by ImClone SystemsIncorporated. Antibodies to growth factors include, IMC-11F8 (similar toERBITUX™), C225 (ERBITUX™), Panitumumab, HERCEPTIN®, IMC-A12 (targetsinsulin-like growth factor-1 receptor (IGF-1R)), IMC-18F1 (targetsvascular endothelial growth factor receptor-1 (VEGFR-1 or flt-1)).Antibodies to angiogensis inhibitors include IMC-1121b (targets vascularendothelial growth factor receptor-2 (VEGFR-2) or Avastin. Antibodies toother cell surface structures include Rituximab (targets CD20).

In another aspect of immunotherapy, the tumor cell must bear some markerthat is amenable to targeting, i.e., is not present on the majority ofother cells. Many tumor markers exist and any of these may be suitablefor targeting in the context of the present invention. Common tumormarkers include carcinoembryonic antigen, prostate specific antigen,urinary tumor associated antigen, fetal antigen, tyrosinase (p97), gp68,TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor,laminin receptor, erb B and p155.

E. Genetic Therapy Agents

A tumor cell resistance to agents, such as chemotherapeutic andradiotherapeutic agents, represents a major problem in clinicaloncology. One goal of current cancer research is to find ways to improvethe efficacy of one or more anti-cancer agents by combining such anagent with gene therapy. For example, the herpes simplex-thymidinekinase (HS-tK) gene, when delivered to brain tumors by a retroviralvector system, successfully induced susceptibility to the antiviralagent ganciclovir (Culver, et al., 1992). In the context of the presentinvention, it is contemplated that gene therapy could be used similarlyin conjunction with the superantigen therapy.

F. Other Anticancer Agents

It is contemplated that other agents may be used in combination with thesuperantigens to improve the therapeutic efficacy of treatment. Theseadditional agents include immunomodulatory agents, agents that affectthe upregulation of cell surface receptors and GAP junctions, cytostaticand differentiation agents, inhibitors of tyrosine kinases includingepidermal growth factor receptor inhibitors, inhibitors of celladhesion, or agents that increase the sensitivity of thehyperproliferative cells to apoptotic inducers. Immunomodulatory agentsinclude tumor necrosis factor; interferon alpha, beta, and gamma; IL-2,linomide, and other cytokines; F42K and other cytokine analogs; orMIP-1, MIP-1beta, MCP-1, RANTES, and other chemokines. It is furthercontemplated that the upregulation of cell surface receptors or theirligands such as Fas/Fas ligand, DR4 or DR5/TRAIL would potentiate theapoptotic inducing abilities of the present invention by establishmentof an autocrine or paracrine effect on hyperproliferative cells.Increases intercellular signaling by elevating the number of GAPjunctions would increase the anti-hyperproliferative effects on theneighboring hyperproliferative cell population.

In other embodiments, cytostatic or differentiation agents can be usedin combination with the present invention to improve theanti-hyperproliferative efficacy of the treatments. Inhibitors oftyrosine kinases include small molecules that act as inhibitors ofepidermal growth factor receptor (EGFR) tyrosine kinase, such as ZD1839(IRESSA®), OSI-774 (TARCEVA™) and passive antibodies (also discussedabove as immune therapy) such as EGFR-blocking antibodies C225(ERBITUX™) ABR-EGF, IMC-11F8, and HERCEPTIN®, split kinase inhibitors,such as PTK787/ZK 222584, SU11248, CP 549,632, and AG013736, and otherinhibitors of tyrosine kinase, such as Imatinib (GLEEVEC®), MLN-518,CEP-701, PKC-412 (see Laird & Cherrington (2003), which is incorporatedherein by reference in its entirety). Inhibitors of cell adhesion arecontemplated to improve the efficacy of the present invention. Examplesof cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitorsand Lovastatin. It is further contemplated that other agents thatincrease the sensitivity of a hyperproliferative cell to apoptosis, suchas the antibody c225, could be used in combination with the presentinvention to improve the treatment efficacy. Other agents includeOSI-7904, Lapatinib, VELCADE®, and Sorafenib.

Hormonal therapy may also be used in conjunction with the presentinvention or in combination with any other cancer therapy previouslydescribed. The use of hormones may be employed in the treatment ofcertain cancers such as breast, prostate, ovarian, or cervical cancer tolower the level or block the effects of certain hormones such astestosterone or estrogen. This treatment can be used in in combinationwith the superantigen therapy of the present invention to reduce therisk of metastases.

V. COMBINATION HYPERPROLIFERATIVE DISEASE THERAPY

The present invention describes the treatment of a hyperproliferativedisease, the reduction of a neoplasm or tumor, and/or the reduction of ametastatic tumor by administering to a subject in need a superantigen incombination with at least one anticancer agent, more preferable achemotherapeutic agent.

A. Types of Hyperproliferative Diseases

The invention may be used in the treatment and prevention ofhyperproliferative diseases including, but not limited to, cancer. Ahyperproliferative disease is any disease or condition which has, aspart of its pathology, an abnormal increase in cell number. Included insuch diseases are benign conditions such as benign prostatic hypertrophyand ovarian cysts. Also included are premalignant lesions, such assquamous hyperplasia. At the other end of the spectrum ofhyperproliferative diseases are cancers. A hyperproliferative diseasecan involve cells of any cell type. The hyperproliferative disease mayor may not be associated with an increase in size of individual cellscompared to normal cells.

Another type of hyperproliferative disease is a hyperproliferativelesion, a lesion characterized by an abnormal increase in the number ofcells. This increase in the number of cells may or may not be associatedwith an increase in size of the lesion. Examples of hyperproliferativelesions that are contemplated for treatment include benign tumors andpremalignant lesions. Examples include, but are not limited to, squamouscell hyperplastic lesions, premalignant epithelial lesions, psoriaticlesions, cutaneous warts, periungual warts, anogenital warts,epidermdysplasia verruciformis, intraepithelial neoplastic lesions,focal epithelial hyperplasia, conjunctival papilloma, conjunctivalcarcinoma, or squamous carcinoma lesion. The lesion can involve cells ofany cell type. Examples include keratinocytes, epithelial cells, skincells, and mucosal cells.

Preneoplastic or hyperplastic states which may be treated or preventedusing the combination treatment of the present invention include but arenot limited to preneoplastic or hyperplastic states such as colonpolyps, Crohn's disease, ulcerative colitis, breast lesions and thelike.

Cancers which may be treated using the pharmaceutical composition of thepresent invention include, but are not limited to primary or metastaticmelanoma, adenocarcinoma, squamous cell carcinoma, adenosquamous cellcarcinoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer,non-Hodgkin's lymphoma, Hodgkin's lymphoma, leukemias, uterine cancer,breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, coloncancer, multiple myeloma, neuroblastoma, NPC, bladder cancer, cervicalcancer and the like. Still further, the cancer that may be treated byadministering a targeted-superantigen and chemotherapeutic agent can bedefined based upon the body location and/or system, for example, but notlimited to bone (e.g., Ewing's Family of tumors, osteosarcoma); brain(e.g., adult brain tumor, (e.g., adult brain tumor, brain stem glioma(childhood), cerebellar astrocytoma (childhood), cerebralastrocytoma/malignant glioma (childhood), ependymoma (childhood).Medulloblastoma (childhood), supratentorial primitive neuroectodermaltumors and pineoblastoma (childhood), visual pathway and hypothalamicglioma (childhood) and childhood brain tumor (other)); breast (e.g.,breast cancer, breast cancer and pregnancy, male breast cancer);digestive/gastrointestinal (e.g., anal cancer, bile duct cancer(extrahepatic), carcinoid tumor (gastrointestinal), colon cancer,esophageal cancer, gallbladder cancer, liver cancer (adult primary),liver cancer (childhood), pancreatic cancer, small intestine cancer,stomach (gastric) cancer); endocrine (e.g., adrenocortical carcinoma,carcinoid tumor (gastrointestinal), islet cell carcinoma (endocrinepancreas), parathyroid cancer, pheochromocytoma, pituitary tumor,thyroid cancer); eye (e.g., melanoma (intraocular), retinoblastoma);genitourinary (e.g., bladder cancer, kidney (renal cell) cancer, penilecancer, prostate cancer, renal plvis and ureter cancer (transitionalcell), testicular cancer, urethral cancer, Wilms' Tumor and otherchildhood kidney tumors); germ cell (e.g., extracranial germ cell tumor(childhood), extragonadal germ cell tumor, ovarian germ cell tumor,testicular cancer); gynecologic (e.g., cervical cancer, endometrialcancer, gestational trophoblastic tumor, ovarian epithelial cancer,ovarian germ cell tumor, ovarian low malignant potential tumor, uterinesarcoma, vaginal cancer, vulvar cancer); head and neck (e.g.,hypopharyngeal cancer, laryngeal cancer, lip and oral cavity cancer,metastatic squamous neck cancer with occult primary, nasopharyngealcancer, oropharyngeal cancer, paranasal sinus and nasal cavity cancer,parathyroid cancer, salivary gland cancer): lung (e.g., non-small celllung cancer, small cell lung cancer); lymphoma (e.g., AIDS-RelatedLymphoma, cutaneous T-cell lymphoma, Hodgkin's Lymphoma (adult),Hodgkin's Lymphoma (childhood), Hodgkin's Lymphoma during pregnancy,mycosis fungoides, Non-Hodgkin's Lymphoma (adult), Non-Hodgkin'sLymphoma (childhood), Non-Hodgkin's Lymphoma during pregnancy, primarycentral nervous system lymphoma, Sezary Syndrome, T-cell lymphoma(cutaneous), Waldenstrom's Macroglobulinemia); musculoskeletal (e.g.,Ewing's Family of tumors, osteosarcoma/malignant fibrous histiocytoma ofbone, rhabdomyosarcoma (childhood), soft tissue sarcoma (adult), softtissue sarcoma (childhood), uterine sarcoma); neurologic (e.g., adultbrain tumor, childhood brain tumor (e.g., brain stem glioma, cerebellarastrocytoma, cerebral astrocytoma/malignant glioma, ependymoma,medulloblastoma, supratentorial primitive neuroectodermal tumors andpineoblastoma, visual pathway and hypothalamic glioma, other braintumors), neuroblastoma, pituitary tumor primary central nervous systemlymphoma); pregnancy and cancer (e.g., breast cancer and pregnancy,Hodgkin's lymphoma during pregnancy, Non-Hodgkin's lymphoma duringpregnancy); respiratory/thoracic (e.g., non-small cell lung cancer,small cell lung cancer, malignant mesothelioma, thymoma and thymiccarcinoma); and skin (e.g., cutaneous T-cell lymphoma, Kaposi's sarcoma,melanoma, merkel cell carcinoma, skin cancer).

Cancers that may be treated using the combination of the presentinvention (targeted-superantigen and a chemotherapeutic agent) may alsobe defined based upon the chemotherapeutic agent that is known to treatthe cancer, for example, but not limited to alkylating agents (e.g.,Hodgkin's disease, lymphomas, and certain carcinomas of the lung,breast, prostate and ovary cancer); nitrosoureas (e.g., brain tumors,lymphomas, multiple myeloma, and malignant melanoma); antimetabolites(e.g., choriocarcinoma, and some tumors of the gastrointestinal tract,breast, lung, kidney and ovary); antitumor antibiotics (e.g., lymphomas,breast, ovarian, endometrium, prostate and gastrointestinal); mitoticinhibitors (e.g., certain gastro-intestinal cancers, Hodgkin's andnon-Hodgkin's lymphomas, neuroblastomas, Wilms' tumor, and cancers ofthe lung, breast, prostate and testes); platinum based compounds (e.g.cancer of the lung, breast, ovary, bladder, head and neck and certaingastrointestinal) and targeted/other therapies (renal, prostate,gastrointestinal, lymphomas, non-hodgkin-lymphoma, breast, lung,myeloma).

Yet further, the cancer may include a tumor comprised of tumor cells.For example, tumor cells may include, but are not limited to melanomacell, a bladder cancer cell, a breast cancer cell, a lung cancer cell, acolon cancer cell, a prostate cancer cell, a liver cancer cell, apancreatic cancer cell, a stomach cancer cell, a testicular cancer cell,a renal cancer cell, an ovarian cancer cell, a lymphatic cancer cell, askin cancer cell, a brain cancer cell, a bone cancer cell, or a softtissue cancer cell.

Other hyperproliferative diseases that may be treated using thecombination treatment of the present invention include, but are notlimited to rheumatoid arthritis, inflammatory bowel disease,osteoarthritis, leiomyomas, adenomas, lipomas, hemangiomas, fibromas,vascular occlusion, restenosis, atherosclerosis, pre-neoplastic lesions(such as adenomatous hyperplasia and prostatic intraepithelialneoplasia), carcinoma in situ, oral hairy leukoplakia, or psoriasis.

In a preferred embodiment of the present invention, the combination ofthe tumor-targeted superantigen and at least one anticancer agent, forexample a cystostatic agent or a radiotherapeutic agent, areadministered in an effective amount to decrease, reduce, inhibit orabrogate the growth of a solid tumor. Examples of solid tumors that canbe treated according to the invention include sarcomas and carcinomassuch as, but not limited to: fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, andretinoblastoma.

Yet further, cancers that are most likely to be treated in the presentinvention are those that metastasize. It is understood by those in theart that metastasis is the spread of cells from a primary tumor to anoncontiguous site, usually via the bloodstream or lymphatics, whichresults in the establishment of a secondary tumor growth. Examplescancers contemplated for treatment include, but are not limited tomelanoma, bladder, non-small cell lung, small cell lung, lung,hepatocarcinoma, retinoblastoma, astrocytoma, glioblastoma,neuroblastoma, head, neck, breast, pancreatic, gum, tongue, prostate,renal, bone, testicular, ovarian, mesothelioma, cervical,gastrointestinal lymphoma, brain, or colon cancer and any other tumorsor neoplasms that may be treated by administering in combination atumor-targeted superantigen and at least one cytostatic agent.

Still further, the present invention can also be used to treat tumors insheaths (“theca”) encasing organs. Examples include (1) pleural effusiondue to fluid in the pleural sheath surrounding the lung, (2) ascitesoriginating from fluid accumulating in the peritoneal membrane and (3)cerebral edema due to metastatic carcinomatosis of the meninges. Sucheffusions and fluid accumulations generally develop at an advanced stageof the disease. Malignant pleural effusion (“MPE”) is the prototype ofthis condition.

B. Therapeutic Amounts

As used herein the terms “effective amount” or “dose” or “therapeuticdose” are defined as an amount of the agent that will decrease, reduce,inhibit or otherwise abrogate the growth or proliferation of a cancercell, induce apoptosis, inhibit angiogenesis of a tumor cell, inhibitmetastasis, or induce cytotoxicity in cells. The effective amount ofactive compound(s) used to practice the present invention fortherapeutic treatment of neoplasms (e.g., cancer) varies depending uponthe manner of administration, the age, body weight, and general healthof the subject. It is within the skill in the art for an attendingphysician or veterinarian to determine the appropriate amount and dosageregimen. Such amounts may be referred to as an “effective” amounts.These terms include synergistic situations such as those presented anddescribed in the instant invention wherein a single agent alone, such asa superantigen or an anticancer agent such as a cytostatic agent, mayact weakly or not at all, but when combined with each other, forexample, but not limited to, via sequential dosage, the two or moreagents act to produce a synergistic result.

In other non-limiting examples, a dose may also comprise from about 1microgram/kg/body weight, about 5 microgram/kg/body weight, about 10microgram/kg/body weight, about 50 microgram/kg/body weight, about 100microgram/kg/body weight, about 200 microgram/kg/body weight, about 350microgram/kg/body weight, about 500 microgram/kg/body weight, about 1milligram/kg/body weight, about 5 milligram/kg/body weight, about 10milligram/kg/body weight, about 50 milligram/kg/body weight, about 100milligram/kg/body weight, about 200 milligram/kg/body weight, about 350milligram/kg/body weight, about 500 milligram/kg/body weight, to about1000 mg/kg/body weight or more per administration, and any rangederivable therein. In non-limiting examples of a derivable range fromthe numbers listed herein, a range of about 5 mg/kg/body weight to about100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500milligram/kg/body weight, etc., can be administered, based on thenumbers described above.

In certain embodiments, for example, but not limited to, TTSadministration, the effective amount or dose of the superantigen that isadministered is an amount in the range of 0.01 to 500 μg/kg body weightof the subject, preferably 0.1-500 μg/kg body weight of the subject, andmost preferably 1-100 μg/kg body weight of the subject. In certainembodiments where wild-type superantigens and/or unconjugated/fusedand/or unmodified superantigens are used, the effective amount or doesof the superantigen that is to be administered is in an amount in therange of 0.001 to 500 μg/kg body weight of the subject.

It is envisioned that the effective amount or dose of the anticanceragent (e.g., a cytostatic agent and/or radiotherapeutic agent) that isadministered in combination with the superantigen is a dose that resultsin a synergistic anti-tumor effect and does not interfere or inhibit theenhancement of the immune system or T-cell activation. Thus, one ofskill in the art is aware that the dose and/or timing of the dose may bealtered depending upon the therapeutic regimen. For example, the dose ofthe anticancer agent (e.g., cytostatic agent and/or radiotherapeuticagent) may be considered a high dose or “anti-proliferative dose” and isadministered prior to and/or subsequent to the superantigenadministration. This type of regiment considers the half-life of theanticancer agent. If the anticancer agent is administered simultaneouslywith the superantigen, then the anticancer agent may be administered ina low dose such that it does not interfere with the mechanism of actionof the superantigen.

C. Treatment Regimens

Treatment regimens may vary as well, and often depend on tumor type,tumor location, disease progression, and health and age of the patient.Obviously, certain types of tumor will require more aggressivetreatment, while at the same time, certain patients cannot tolerate moretaxing protocols. The clinician may often be best suited to make suchdecisions based on his or her skill in the art and the known efficacyand toxicity (if any) of the therapeutic formulations.

Preferably, patients to be treated will have adequate bone marrowfunction (defined as a peripheral absolute granulocyte count of>2,000/mm³ and a platelet count of 100,000/mm³), adequate liver function(bilirubin<1.5 mg/dl) and adequate renal function (creatinine<1.5mg/dl).

To kill cells, inhibit cell growth, inhibit metastasis, decrease tumoror tissue size and otherwise reverse or reduce the malignant phenotypeof tumor cells, using the methods and compositions of the presentinvention, one would generally contact a tumor cell or neoplasm orcancer cell with a combination of a superantigen and at least oneanticancer agent, such as a cytostatic agent and/or a radiotherapeuticagent. The routes of administration will vary, naturally, with thelocation and nature of the lesion, and include, e.g., intradermal,transdermal, parenteral, intravenous, intrathecal, intramuscular,intranasal, subcutaneous, percutaneous, intratracheal, intraperitoneal,intratumoral, perfusion, lavage, direct injection, and oraladministration and formulation.

In one aspect of the present invention, the tumor cell or neoplasm orcancer cell must bear some marker that is amenable to targeting, e.g.,is not present on the majority of other cells. Many tumor markers existand any of these may be suitable for targeting in the context of thepresent invention. The selection of a suitable marker is will within theskill of one in the art and skill in the related art. Specific targetingagents of the present invention include, e.g., antibodies. Theantibodies that are contemplated in the present invention include, butare not limited to the Fab fragment. Examples of the Fab fragmentinclude C215Fab or 5T4Fab. In addition to Fab, other common tumormarkers include carcinoembryonic antigen, prostate specific antigen,urinary tumor associated antigen, fetal antigen, tyrosinase (p97), gp68,TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor,laminin receptor, erb B and p155.

In certain embodiments, the treatment regimen of the present inventionmay involve contacting the neoplasm or tumor cells with the superantigenand the cytostatic agent at the same time. This may be achieved bycontacting the cell with a single composition or pharmacologicalformulation that includes both agents, or by contacting the cell withtwo distinct compositions or formulations, at the same time, wherein onecomposition includes the superantigen and the other includes theanticancer agent, such as a cytostatic agent.

Alternatively, the superantigen of the present invention may precede orfollow the anticancer agent by intervals ranging from minutes, days toweeks. In embodiments where the other anticancer agent and thesuperantigen are applied separately to the cell, one would generallyensure that a significant period of time did not expire between the timeof each delivery, such that the superantigen and anticancer agent wouldstill be able to exert an advantageously combined effect on the cell. Insuch instances, it is contemplated that one may contact the cell withboth modalities within about 12-72 h of each other. In some situations,it may be desirable to extend the time period for treatmentsignificantly, however, where several days (2, 3, 4, 5, 6 or 7) toseveral weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respectiveadministrations.

Various combinations may be employed, the superantigen is “A” and theanticancer agent, is “B”:

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B;B/A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A;B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A; and A/A/B/A.

The terms “contacted” and “exposed,” as used herein means when appliedto a cell, are used herein to describe the process by which thesuperantigen and a cytostatic agent or a radiotherapeutic agent aredelivered to a target cell or are placed in direct juxtaposition withthe target cell. To achieve cell killing or stasis, both agents aredelivered to a cell in a combined amount effective to kill the cell orprevent it from dividing. This delivery may be sequentially timed asdescribed herein.

Timing of the therapeutic regimen may be related to the half-live of theanticancer agent, such as a cytostatic agent. Half-life of a cytostaticagent can be determined using the below equationHalf-Life=(0.693×V _(d))/CL

CL is defined as the clearance of the agent or drug clearance, which isthe volume of plasma cleared of drug per unit time. V_(d) is defined asthe volume of distribution, which is the amount or concentration of theagent in the plasma or blood to the total amount in the body. Thehalf-life can be used to determine the timing for sequentialadministration of a cytostatic agent followed by superantigen therapy.Thus, in practicing the present invention, one skilled in the art wouldknow that superantigen therapy could be sequentially administered incombination with a cytostatic, for example, by first treating with thecytostatic agent. Once the effective cytostatic concentration of agenthas dropped in the patient below a functional inhibitory level basedupon the half-life of the agent, the superantigen can be administered tothe patient.

It is further envisioned that the present invention can be used incombination with surgical intervention. In the case of surgicalintervention, the present invention may be used preoperatively, e.g., torender an inoperable tumor subject to resection. Alternatively, thepresent invention may be used at the time of surgery, and/or thereafter,to treat residual or metastatic disease. For example, a resected tumorbed may be injected or perfused with a formulation comprising thetumor-targeted superantigen and/or the anticancer agent (e.g., acytostatic agent or a radiotherapeutic agent). The perfusion may becontinued post-resection, for example, by leaving a catheter implantedat the site of the surgery. Periodic post-surgical treatment also isenvisioned. Any combination of the invention therapy with surgery iswithin the scope of the invention.

Continuous administration also may be applied where appropriate, forexample, where a tumor is excised and the tumor bed is treated toeliminate residual, microscopic disease. Delivery via syringe orcatherization is preferred. Such continuous perfusion may take place fora period from about 1-2 hours, to about 2-6 hours, to about 6-12 hours,to about 12-24 hours, to about 1-2 days, to about 1-2 wk or longerfollowing the initiation of treatment. Generally, the dose of thetherapeutic composition via continuous perfusion will be equivalent tothat given by a single or multiple injections, adjusted over a period oftime during which the perfusion occurs. It is further contemplated thatlimb perfusion may be used to administer therapeutic compositions of thepresent invention, particularly in the treatment of melanomas andsarcomas.

In certain embodiments, the tumor being treated may not, at leastinitially, be resectable. Treatments with superantigen in combinationwith at least one anticancer agent may increase the resectability of thetumor due to shrinkage at the margins or by elimination of certainparticularly invasive portions. Following treatments, resection may bepossible. Additional treatments subsequent to resection will serve toeliminate microscopic residual disease at the tumor site.

A typical course of treatment, for a primary tumor or a post-excisiontumor bed, may involve multiple doses. Typical primary tumor treatmentmay involve a 6 dose application over a two-week period. The two-weekregimen may be repeated one, two, three, four, five, six or more times.During a course of treatment, the need to complete the planned dosingsmay be re-evaluated.

The treatments may include various “unit doses.” Unit dose is defined ascontaining a predetermined-quantity of the therapeutic composition. Thequantity to be administered, and the particular route and formulation,are within the skill of those in the clinical arts. A unit dose need notbe administered as a single injection but may comprise continuousinfusion over a set period of time.

As used in the present invention, a combination treatment regimenconsists of administering a superantigen and at least one anticanceragent, for example a cytostatic drug or a radiotherapeutic agent. Yetfurther, it may be desirable to combine the combination treatmentregimen with other agents effective in the treatment of neoplasticdiseases or cancers, such as other anticancer agents, such as otherinclude biological agents (biotherapy) and/or hormonal therapy, etc.

Included within the scope of the present invention is the systemic(e.g., via intravenous (iv) administration) of superantigens, forexample TTS, and/or anti-cancer agents, for example chemotherapeuticdrugs, for example cytostatic drugs. Also included is the localadministration (e.g., via direct administration of the agent to thetumor, for example by intrathecal administration) of superantigens, forexample TTS, and/or anti-cancer agents, for example chemotherapeuticdrugs, for example cytostatic drugs. Also included is the combination oflocal and systemic administration of superantigens, for example TTS,and/or anti-cancer agents, for example chemotherapeutic drugs, forexample cytostatic drugs (for example, systemic administration ofsuperantigens (e.g., TTS) combined with local administration of ananti-cancer agent (e.g., a chemotherapeutic drug) and vice versa).

VI. COMBINED THERAPY

As used herein, the term “sequential dosage” and related terminologyrefers to the administration of at least one superantigen, with at leastone anticancer agent, for example, but not limited to a chemotherapeuticagent, such as but not limited to a chemotherapeutic drug, such as butnot limited to cytostatic chemotherapeutic drug. This definitionincludes staggered doses of these agents and variations in dosageamounts. This includes one agent being administered before, overlappingwith (partially or totally), after, or totally separate from anotheragent. This term generally considers the best administration scheme toachieve a synergistic combination of at least one superantigen and atleast one anticancer agent and/or to achieve administration of at leastone superantigen while limiting or eliminating the generation of anantibody response to the superantigen. Determining sequential dosageadministration plans is within the skill of one in the art, from thebackground skill and teaching in the art and the teaching of thisapplication. For example, one of skill in the art, e.g., based on knowndrug half-lifes, is able to determine when an anticancer agent orcytostatic agent is below a functional inhibitory level. A functionalinhibitory level of the anti-cancer agent is determined based upon theagent's half-life. Thus, a skilled artisan would know that in order forthe superantigen therapy to be effective it must be administered afterthe cytostatic agent is below a functional inhibitory level.

Typically, one of skill in the art considers chemotherapy andradiotherapy to have anti-proliferative properties, and therefore wouldgenerally be expected to interfere with immune stimulating agents suchas tumor vaccines, biological response modifiers and superantigens. Thepresent invention utilizes the combination of these knownanti-proliferative therapies in combination with a known immunestimulatory or proliferative agent.

It is well documented that certain drugs may also haveimmune-stimulatory properties when administered at low (sub-toxic) doses(reviewed by Zagozdzon and Golab, 2001; Mitchell, 2003). However, theinventors of the present invention have provided an intergrated highdose cytostatic agent/immunotherapy treatment. The prerequisites of anintegrated high dose cytostatic drug/immunotherapy treatment relate todistinct time frames for the activity of the agents. The cytostaticagent should act within a certain time frame and the same is necessaryfor the immunotherapy.

Most cytostatic drugs act during a short period of high drug exposure,and elimination of the drug generally occurs within 24 hours afteradministration. Typically, serum half-lifes of the commonly used drugsgemcitabine and docetaxel are 42-94 minutes and 11.1 hours respectively.Other half-lifes and half-live determinations are discussed above, whichis incorporated herein.

The immunotherapy though is generally dependent on a long period ofimmune activation with unpredicted phases of lymphocyte proliferation.Thus, the initiation of an antigen specific immune response, e.g.,during vaccination, depends on a number of time limiting processes, suchas targeting of antigen to lymphoid organs, uptake and presentation onantigen presenting cells etc. Also, the duration of the response mayvary considerably, depending on the persistency of antigen in thecirculation.

The immunotherapy of superantigens results in rapid (within hours) andpowerful polyclonal activation of T lymphocytes. A superantigentreatment cycle commonly includes 4 to 5 daily intravenous superantigendrug injections. Such treatment cycles can be given in e.g., 4 to 6weeks intervals. Non-clinical studies show a nearly instant T lymphocyteactivation and proliferation and rapid inflammation by cytotoxic Tlymphocytes (CTLs) into tumor tissue as a response to the firstinjection of the superantigen drug (Dohlsten et al, 1995). Theinflammation with infiltration of CTLs into the tumor is one of themajor effectors of the anti-tumor therapeutic superantigens. After ashort period of massive activation and differentiation of CTLs, theT-cell response declines rapidly (within 4-5 days) back to base linelevels. Thus, the period of lymphocyte proliferation, during whichcytostatic drugs may interfere with superantigen treatment is short andwell-defined. Only with the superantigen/anti-cancer agent therapy ofthe instant invention is such a distinct time frame for activityplausible, thereby allowing the novel integrated high dose cytostaticagent/immunotherapy treatment.

The cytostatic drugs and the immunotherapy superantigens represent typesof anti-tumor therapies with distinct mechanisms of action potentiallynot possible to timely integrate and should therefore not be additive.Surprisingly though, by combining high dose cytostatic drugs withsuperantigens in integrated treatment schedules synergistic anti-tumoreffects are obtained. Thus, in a general combination therapy schedulesuperantigen treatment may be integrated with one or more standardchemotherapy (at well established standard doses) in such a way thatsuperantigen is given shortly (e.g., within 0-3 days or, e.g., 0-6 days)before or after administration of the cytostatic drug.

Administration of the immunotherapy of the present invention to apatient will follow general protocols for the administration ofchemotherapeutics. It is expected that the treatment cycles would berepeated as necessary. It also is contemplated that various standardtherapies, as well as surgical intervention, may be applied incombination with the described neoplastic cell therapy.

In addition to the combination of the superantigen and cytostatic agenthaving a synergistic anti-tumor effect, the cystostatic agent modulatesor inhibits production of antibodies to superantigens

The superantigen fusion proteins are immunogenic and antibodies areproduced. These bind to the superantigen protein and are an obstacle forsuperantigen activity. Primary antibody responses are not productiveuntil after about one week post stimulation. Secondary antibodyresponses usually have their maximal B cell proliferative phaseapproximately 5-7 days after stimulation.

Antibodies interfering with the superantigen proteins compromiseanti-tumor therapeutic effects. Therefore the instant combinationtreatment that results in no, or fewer anti-superantigen antibodies in apatient makes multi-cycle superantigen treatment possible.

VII. KITS

Embodiments of the present invention include kits comprising, forexample, a first container having a tumor-targeted superantigen and asecond container having at least one anticancer agent, such as achemotherapeutic agent, such as a cytostatic agent.

Any of the compositions described herein may be comprised in a kit. In anon-limiting example, a kit may comprise a superantigen (for example, aTTS) and a cytostatic agent. Such a kit may also contain additionalagents such as, for example, a lipid component.

The kits may comprise a suitably aliquoted a superantigen and/or acytostatic agent, and optionally, lipid and/or additional agentcompositions of the present invention. The components of the kits may bepackaged either in aqueous media or in lyophilized form. The containermeans of the kits will generally include at least one vial, test tube,flask, bottle, syringe or other container means, into which a componentmay be placed, and preferably, suitably aliquoted. Where there are morethan one component in the kit, the kit also will generally contain asecond, third or other additional container into which the additionalcomponents may be separately placed. However, various combinations ofcomponents may be comprised in one or more vials. The kits of thepresent invention also will typically include a means for containing thea superantigen and/or a cytostatic agent, lipid, additional agent, andany other reagent containers in close confinement for commercial sale.Such containers may include injection or blow molded plastic containersinto which the desired vials are retained.

Therapeutic kits of the present invention will generally contain, insuitable container means, a pharmaceutically acceptable formulation of asuperantigen and an anti-cancer agent in a pharmaceutically acceptableformulation. The kit may have a single container means, and/or it mayhave distinct container means for each compound.

When the components of the kit are provided in one and/or more liquidsolutions, the liquid solution is an aqueous solution, with a sterileaqueous solution being particularly preferred. The a superantigen and ananti-cancer agent may also be formulated into a syringeable composition.In which case, the container means may itself be a syringe, pipette,and/or other such like apparatus, from which the formulation may beapplied to a specific area of the body, injected into an animal, and/orapplied and/or mixed with the other components of the kit.

The components of the kit may be provided as dried powder(s). Whenreagents and/or components are provided as a dry powder, the powder canbe reconstituted by the addition of a suitable solvent. It is envisionedthat the solvent may also be provided in another container means.

The container will generally include at least one vial, test tube,flask, bottle, syringe and/or other type for holding a sample, intowhich the a superantigen, and/or an anti-cancer agent are placed, e.g.,suitably allocated. The kits may also comprise another container forcontaining a sterile, pharmaceutically acceptable buffer and/or otherdiluent.

The kits of the invention may also comprise, and/or be packaged with, aninstrument for assisting with the injection/administration and/orplacement of the ultimate therapeutic composition, e.g., a superantigenand a cytostatic agent, within the body of an animal. Such an instrumentmay be a syringe, pipette, forceps, and/or any such medically approveddelivery vehicle.

VIII. PHARMACEUTICAL COMPOSITIONS

The combined superantigen and anti-cancer agents of the presentinvention may be employed alone or in conjunction with other compounds,such as carriers or other therapeutic compounds.

Pharmaceutical compositions of the present invention comprise aneffective amount of one or more superantigens and one or moreanti-cancer agents, for example a chemotherapeutic agent, such as acytostatic agent, and may also contain additional agents, dissolved ordispersed in a pharmaceutically acceptable carrier. The phrases“pharmaceutical or pharmacologically acceptable” refer to substances,e.g., compositions, that do not produce an adverse, allergic or otheruntoward reaction when administered to a mammal, such as, for example, ahuman. The preparation of a pharmaceutical composition that contains atleast one superantigen and an anti-cancer agent will be known to thoseof skill in the art in light of the present disclosure, and asexemplified by Remington's Pharmaceutical Sciences, 18th Ed. MackPrinting Company, 1990, incorporated herein by reference. Moreover, foranimal (e.g., human) administration, it will be understood thatpreparations should meet sterility, pyrogenicity, general safety andpurity standards as required by FDA Office of Biological Standards.

As used herein, “pharmaceutically acceptable carrier” includes, e.g.,solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated hereinby reference). Except insofar as any conventional carrier isincompatible with the active ingredient, its use in the pharmaceuticalcompositions is contemplated.

The superantigens and anti-cancer agents may comprise different types ofcarriers depending on whether they are to be administered in solid,liquid or aerosol form, and whether it need to be sterile for suchroutes of administration as injection. The present invention maypossibly be administered intravenously, intradermally, transdermally,intrathecally, intraarterially, intraperitoneally, intranasally,intravaginally, intrarectally, topically, intramuscularly,subcutaneously, mucosally, orally, topically, locally, inhalation (e.g.,aerosol inhalation), injection, infusion, continuous infusion, localizedperfusion bathing target cells directly, via a catheter, via a lavage,in cremes, in lipid compositions (e.g., liposomes), or by other methodor any combination of the forgoing as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed. Mack Printing Company, 1990, incorporated herein by reference).

The superantigens and anti-cancer agent may be formulated into acomposition in a free base, neutral or salt form. Pharmaceuticallyacceptable salts include the acid addition salts, e.g., those formedwith the free amino groups of a proteinaceous composition, or which areformed with inorganic acids such as for example, hydrochloric orphosphoric acids, or such organic acids as acetic, oxalic, tartaric ormandelic acid. Salts formed with the free carboxyl groups can also bederived from inorganic bases such as for example, sodium, potassium,ammonium, calcium or ferric hydroxides; or such organic bases asisopropylamine, trimethylamine, histidine or procaine. Upon formulation,solutions will be administered in a manner compatible with the dosageformulation and in such amount as is therapeutically effective. Theformulations are easily administered in a variety of dosage forms suchas formulated for parenteral administrations such as injectablesolutions, or aerosols for delivery to the lungs, or formulated foralimentary administrations such as drug release capsules and the like.

Further in accordance with the present invention, the composition of thepresent invention suitable for administration is provided in apharmaceutically acceptable carrier with or without an inert diluent.The carrier should be assimilable and includes liquid, semi-solid, e.g.,pastes, or solid carriers. Except insofar as any conventional media,agent, diluent or carrier is detrimental to the recipient or to thetherapeutic effectiveness of a the composition contained therein, itsuse in administrable composition for use in practicing the methods ofthe present invention is appropriate. Examples of carriers or diluentsinclude fats, oils, water, saline solutions, lipids, liposomes, resins,binders, fillers and the like, or combinations thereof. The compositionmay also comprise various antioxidants to retard oxidation of one ormore component. Additionally, the prevention of the action ofmicroorganisms can be brought about by preservatives such as variousantibacterial and antifungal agents, including but not limited toparabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol,sorbic acid, thimerosal or combinations thereof.

In accordance with the present invention, the composition is combinede.g., a carrier in any convenient and practical manner, e.g., bysolution, suspension, emulsification, admixture, encapsulation,absorption and the like. Such procedures are routine for those skilledin the art.

In a specific embodiment of the present invention, the composition iscombined and/or mixed thoroughly with a semi-solid or solid carrier. Themixing can be carried out in any convenient manner such as grinding.Stabilizing agents can be also added in the mixing process in order toprotect the composition from loss of therapeutic activity, e.g.,denaturation in the stomach. Examples of stabilizers for use in an thecomposition include buffers, amino acids such as glycine and lysine,carbohydrates such as dextrose, mannose, galactose, fructose, lactose,sucrose, maltose, sorbitol, mannitol, etc.

In further embodiments, the present invention may concern the use of apharmaceutical lipid vehicle compositions that include may superantigensand/or an anti-cancer agent, as well as, one or more lipids, and anaqueous solvent. As used herein, the term “lipid” will be defined toinclude any of a broad range of substances that are characteristicallyinsoluble in water and extractable with an organic solvent. This broadclass of compounds are well known to those of skill in the art, and asthe term “lipid” is used herein, it is not limited to any particularstructure. Examples include compounds which contain long-chain aliphatichydrocarbons and their derivatives. A lipid may be naturally occurringor synthetic (e.g., designed or produced by man). However, a lipid isusually a biological substance. Biological lipids are well known in theart, and include for example, neutral fats, phospholipids,phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids,glycolipids, sulphatides, lipids with ether and ester-linked fatty acidsand polymerizable lipids, and combinations thereof. Of course, compoundsother than those specifically described herein that are understood byone of skill in the art as lipids are also encompassed by thecompositions and methods of the present invention.

Those of ordinary skill in the art are familiar with the range oftechniques that can be employed for dispersing a composition in a lipidvehicle. For example, the superantigens and/or a anti-cancer agents maybe dispersed in a solution containing a lipid, dissolved with a lipid,emulsified with a lipid, mixed with a lipid, combined with a lipid,covalently bonded to a lipid, contained as a suspension in a lipid,contained or complexed with a micelle or liposome, or otherwiseassociated with a lipid or lipid structure by any means known to thoseof ordinary skill in the art. The dispersion may or may not result inthe formation of liposomes.

The actual dosage amount of a composition of the present inventionadministered to an animal patient can routinely be determined by one ofskill in the art by physical and physiological factors such as bodyweight, severity of condition, the type of disease being treated,previous or concurrent therapeutic interventions, idiopathy of thepatient and on the route of administration. Depending upon the dosageand the route of administration, the number of administrations of apreferred dosage and/or an effective amount may vary according to theresponse of the subject. The practitioner responsible for administrationwill, in any event, determine the concentration of active ingredient(s)in a composition and appropriate dose(s) for the individual subject.Such determinations are known and used by those of skill in the art.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of an active compound. In otherembodiments, the an active compound may comprise between about 2% toabout 75% of the weight of the unit, or between about 25% to about 60%,for example, and any range derivable therein. Naturally, the amount ofactive compound(s) in each therapeutically useful composition may beprepared is such a way that a suitable dosage will be obtained in anygiven unit dose of the compound. Factors such as solubility,bioavailability, biological half-life, route of administration, productshelf life, as well as other pharmacological considerations will becontemplated by one skilled in the art of preparing such pharmaceuticalformulations, and as such, a variety of dosages and treatment regimensmay be desirable. Such determinations are known and used by those ofskill in the art.

A. Alimentary Compositions and Formulations

In some embodiments of the present invention, the superantigens and/oran anti-cancer agent such as a chemotherapeutic agent, such as acytostatic drug may be formulated to be administered via an alimentaryroute. Alimentary routes include all possible routes of administrationin which the composition is in direct contact with the alimentary tract.Specifically, the pharmaceutical compositions disclosed herein may beadministered orally, buccally, rectally, or sublingually. As such, thesecompositions may be formulated with an inert diluent or with anassimilable edible carrier, or they may be enclosed in hard- orsoft-shell gelatin capsule, or they may be compressed into tablets, orthey may be incorporated directly with the food of the diet.

In certain embodiments, the active compounds may be incorporated withexcipients and used in the form of ingestible tablets, buccal tables,troches, capsules, elixirs, suspensions, syrups, wafers, and the like(U.S. Pat. Nos. 5,641,515; 5,580,579 and 5,792,451, each specificallyincorporated herein by reference in its entirety). The tablets, troches,pills, capsules and the like may also contain the following: a binder,such as, for example, gum tragacanth, acacia, cornstarch, gelatin orcombinations thereof; an excipient, such as, for example, dicalciumphosphate, mannitol, lactose, starch, magnesium stearate, sodiumsaccharine, cellulose, magnesium carbonate or combinations thereof; adisintegrating agent, such as, for example, corn starch, potato starch,alginic acid or combinations thereof; a lubricant, such as, for example,magnesium stearate; a sweetening agent, such as, for example, sucrose,lactose, saccharin or combinations thereof; a flavoring agent, such as,for example peppermint, oil of wintergreen, cherry flavoring, orangeflavoring, etc. When the dosage unit form is a capsule, it may contain,in addition to materials of the above type, a liquid carrier. Variousother materials may be present as coatings or to otherwise modify thephysical form of the dosage unit. For instance, tablets, pills, orcapsules may be coated with shellac, sugar, or both. When the dosageform is a capsule, it may contain, in addition to materials of the abovetype, carriers such as a liquid carrier. Gelatin capsules, tablets, orpills may be enterically coated. Enteric coatings prevent denaturationof the composition in the stomach or upper bowel where the pH is acidic.See, e.g., U.S. Pat. No. 5,629,001. Upon reaching the small intestines,the basic pH therein dissolves the coating and permits the compositionto be released and absorbed by specialized cells, e.g., epithelialenterocytes and Peyer's patch M cells. A syrup of elixir may contain theactive compound sucrose as a sweetening agent methyl and propylparabensas preservatives, a dye and flavoring, such as cherry or orange flavor.Of course, any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic in the amountsemployed. In addition, the active compounds may be incorporated intosustained-release preparation and formulations.

For oral administration the compositions of the present invention mayalternatively be incorporated with one or more excipients in the form ofa mouthwash, dentifrice, buccal tablet, oral spray, or sublingualorally-administered formulation. For example, a mouthwash may beprepared incorporating the active ingredient in the required amount inan appropriate solvent, such as a sodium borate solution (Dobell'sSolution). Alternatively, the active ingredient may be incorporated intoan oral solution such as one containing sodium borate, glycerin andpotassium bicarbonate, or dispersed in a dentifrice, or added in atherapeutically-effective amount to a composition that may includewater, binders, abrasives, flavoring agents, foaming agents, andhumectants. Alternatively the compositions may be fashioned into atablet or solution form that may be placed under the tongue or otherwisedissolved in the mouth.

Additional formulations which are suitable for other modes of alimentaryadministration include suppositories. Suppositories are solid dosageforms of various weights and shapes, usually medicated, for insertioninto the rectum. After insertion, suppositories soften, melt or dissolvein the cavity fluids. In general, for suppositories, traditionalcarriers may include, for example, polyalkylene glycols, triglyceridesor combinations thereof. In certain embodiments, suppositories may beformed from mixtures containing, for example, the active ingredient inthe range of about 0.5% to about 10%, and preferably about 1% to about2%.

B. Parenteral Compositions and Formulations

In further embodiments, superantigens and/or an anti-cancer agent (e.g.,a chemotherapeutic agent, such as a cytostatic drug) may be administeredvia a parenteral route. As used herein, the term “parenteral” includesroutes that bypass the alimentary tract. Specifically, thepharmaceutical compositions disclosed herein may be administered forexample, but not limited to intravenously, intradermally,intramuscularly, intraarterially, intrathecally, intratumorally,subcutaneous, or intraperitoneally U.S. Pat. Nos. 6,613,308, 5,466,468,5,543,158; 5,641,515; 5,399,363 and International Publication WO03094846(each specifically incorporated herein by reference in its entirety).

Solutions of the active compounds as free base or pharmacologicallyacceptable salts may be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions may also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms. The pharmaceutical forms suitable for injectable useinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersions (U.S. Pat. No. 5,466,468, specifically incorporated hereinby reference in its entirety). In all cases the form must be sterile andmust be fluid to the extent that easy injectability exists. It must bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (e.g., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous, and intraperitoneal administration. In thisconnection, sterile aqueous media that can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biologics standards.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. A powdered composition is combined with a liquidcarrier such as, e.g., water or a saline solution, with or without astabilizing agent.

C. Miscellaneous Pharmaceutical Compositions and Formulations

In other embodiments of the invention, superantigens and/or ananti-cancer agent (e.g, a chemotherapeutic agent, such as a cytostaticdrug) may be formulated for administration via various miscellaneousroutes, for example, topical (e.g., transdermal) administration, mucosaladministration (intranasal, vaginal, etc.) and/or inhalation.

Pharmaceutical compositions for topical administration may include theactive compound formulated for a medicated application such as anointment, paste, cream or powder. Ointments include all oleaginous,adsorption, emulsion and water-solubly based compositions for topicalapplication, while creams and lotions are those compositions thatinclude an emulsion base only. Topically administered medications maycontain a penetration enhancer to facilitate adsorption of the activeingredients through the skin. Suitable penetration enhancers includeglycerin, alcohols, alkyl methyl sulfoxides, pyrrolidones andluarocapram. Possible bases for compositions for topical applicationinclude polyethylene glycol, lanolin, cold cream and petrolatum as wellas any other suitable absorption, emulsion or water-soluble ointmentbase. Topical preparations may also include emulsifiers, gelling agents,and antimicrobial preservatives as necessary to preserve the activeingredient and provide for a homogenous mixture. Transdermaladministration of the present invention may also comprise the use of a“patch.” For example, the patch may supply one or more active substancesat a predetermined rate and in a continuous manner over a fixed periodof time.

In certain embodiments, the pharmaceutical compositions may be deliveredby eye drops, intranasal sprays, inhalation, and/or other aerosoldelivery vehicles. Methods for delivering compositions directly to thelungs via nasal aerosol sprays has been described e.g., in U.S. Pat.Nos. 5,756,353 and 5,804,212 (each specifically incorporated herein byreference in its entirety). Likewise, the delivery of drugs usingintranasal microparticle resins (Takenaga et al., 1998) andlysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871,specifically incorporated herein by reference in its entirety) are alsowell-known in the pharmaceutical arts. Likewise, transmucosal drugdelivery in the form of a polytetrafluoroetheylene support matrix isdescribed in U.S. Pat. No. 5,780,045 (specifically incorporated hereinby reference in its entirety).

The term aerosol refers to a colloidal system of finely divided solid ofliquid particles dispersed in a liquefied or pressurized gas propellant.The typical aerosol of the present invention for inhalation will consistof a suspension of active ingredients in liquid propellant or a mixtureof liquid propellant and a suitable solvent. Suitable propellantsinclude hydrocarbons and hydrocarbon ethers. Suitable containers willvary according to the pressure requirements of the propellant.Administration of the aerosol will vary according to subject's age,weight and the severity and response of the symptoms.

IX. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Tumor Targeted Superantigens

The fusion protein C215Fab-SEA (SEQ. ID. NO. 5) (sometimes referred toherein as “ABR-214720”), consisting of Staphylococcal Enterotoxin Agenetically fused to the Fab moiety of an antibody (C215) recognizing anepitope of the human tumor associated antigen EpCAM/GA733-2, wasexpressed in E. coli (Dohlsten et. al., 1994) and purified as describedby Forsberg et al., 1997. Other fusion proteins that may be used include5T4Fab-SEA/E-120 (SEQ. ID. NO. 7) (sometimes referred to herein as“ABR-217620”), and 5T4Fab-SEA_(D227A) (SEQ. ID. NO. 6), which areproduced as described by Forsberg et al., 1997.

Example 2 TTS-Chemotherapeutic (e.g., Gemcitabine, Docetaxel, Cisplatin)Agent Combination Therapy: In Vitro Anti-Tumor

The effect of superantigen (TTS) treatment (5T4Fab-SEA/E-120) incombination with cytostatics on tumor growth was assessed in vitro.

Cell Lines and Media

The human cancer cell line Colo 205 was cultured in RPMI 1640+10% FBS(R10 medium). The cells were prepared by detachment with celldissociation solution, washed with PBS and resuspended in R10 medium.Peripheral blood mononuclear cells (PBMC) were obtained from blooddonors. The PBMC were isolated by density centrifugation onFicoll-Paque. The cells were then cultured in R10 medium and stimulatedwith TTS to obtain effector T-cell lines.

Inhibition of Tumor Growth

Tumor growth inhibition by TTS in combination with gemcitabine(GEMZAR®), docetaxel (TAXOTERE®), and cisplatin was measured by a tumorcell proliferation assay. The proliferation assay measures DNA synthesisof the tumor cells by incorporation of ³H-thymidine. The effector Tcells were prevented from proliferating by pre-treatment of Mitomycin C.Tumor cells were first incubated with TTS and effector T cells for 4hours in vitro. Cytostatics were then added for a total incubation timeof 48 hours, followed by measurements of incorporated radioactivethymidine by liquid scintillation. The background level of incorporatedradioactivity from wells of Mitomycin C-treated T cells alone (<1% ofthe levels in cultures of untreated tumor cells) was subtracted.

${\%\mspace{14mu}{of}\mspace{14mu}{control}} = {\frac{{{experimental}\mspace{14mu}{value}} - {background}}{{{untreated}\mspace{14mu}{tumor}\mspace{14mu}{cells}} - {background}} \times 100}$Results

Treatment of the human Colo 205 tumor cells with activated T cells andABR-217620 in the combination with gemcitabine (GEMZAR®), docetaxel(TAXOTERE®) or cisplatin as applied subsequent to the SADCC(Superantigen Antibody Dependent Cell mediated cytotoxicity) resulted inenhanced inhibition of tumor cell growth. Tables 2-4 depictproliferation of tumor cells as % of untreated control. Treatment withgemcitabine (GEMZAR®), docetaxel (TAXOTERE®) or cisplatin showedenhanced tumor growth inhibition in the combination with ABR-217620.

TABLE 2 Gemcitabine (GEMZAR ®) enhances tumor growth inhibition in thecombination with ABR-217620. Effect on human tumor cell (Colo 205)proliferation, SADCC, 4 h Chemo, 48 h Percent of control Vehicle Vehicle100 ABR-217620, Vehicle 47 1 pM Vehicle Gemcitabine, 16 10 ng/mlABR-217620, Gemcitabine, 7 1 pM 10 ng/ml

TABLE 3 Docetaxel (TAXOTERE ®) enhances tumor growth inhibition in thecombination with ABR-217620. Effect on human tumor cell (Colo 205)proliferation, SADCC, 4 h Chemo, 48 h Percent of control Vehicle Vehicle100 ABR-217620, Vehicle 47 1 pM Vehicle Docetaxel, 46 1 ng/mlABR-217620, Docetaxel, 28 1 pM 1 ng/ml

TABLE 4 Cisplatin enhances tumor growth inhibition in the combinationwith ABR-217620. Effect on human tumor cell (Colo 205) proliferation,SADCC, 4 h Chemo, 48 h Percent of control Vehicle Vehicle 100ABR-217620, Vehicle 47 1 pM Vehicle Cisplatin, 25 1 μg/ml ABR-217620,Cisplatin, 10 1 pM 1 μg/ml

The above results showed that the combination of the chemotherapeuticagents gemcitabine (GEMZAR®), docetaxel (TAXOTERE®) or cisplatin withTTS enhanced the tumor growth inhibition induced by TTS.

Example 3 TTS-Chemotherapeutic Agent (e.g., Pemetrexed) CombinationTherapy In Vitro Anti-Tumor

The effect of superantigen (TTS) treatment (5T4Fab-SEA/E-120) incombination with cytostatics on tumor growth was assessed in vitro.

Cell Lines and Media

The human cancer cell line Colo 205 was cultured in RPMI 1640+10% FBS(R10 medium). The cells were prepared by detachment with celldissociation solution, washed with PBS and resuspended in R10 medium.Peripheral blood mononuclear cells (PBMC) were obtained from blooddonors. The PBMC were isolated by density centrifugation onFicoll-Paque. The cells were then cultured in R10 medium and stimulatedwith TTS to obtain effector T-cell lines.

Inhibition of Tumor Growth

Tumor growth inhibition by TTS and pemetrexed (ALIMTA®) and thecombination was measured by a cell viability test. The cell viabilitytest measured the amount of ATP in the tumor cells using aluciferase-based assay. Tumor cells were first incubated with TTS andeffector T cells for 4 hours in vitro. Pemetrexed was then added for atotal incubation time of 48 hours, followed by measurements of the ATPcontent in the tumor cell cultures. The background level of ATP fromwells of Mitomycin C-treated T cells alone (≦1% of the levels incultures of untreated tumor cells) was subtracted.

${\%\mspace{14mu}{of}\mspace{14mu}{control}} = {\frac{{{experimental}\mspace{14mu}{value}} - {background}}{{{untreated}\mspace{14mu}{tumor}\mspace{14mu}{cells}} - {background}} \times 100}$Results

Treatment of the human Colo 205 tumor cells with activated T cells andABR-217620 in the combination with pemetrexed (ALIMTA®) as appliedsubsequent to the SADCC (Superantigen Antibody Dependent Cell-mediatedCytotoxicity) resulted in enhanced inhibition of tumor cell growth.Table 5 depicts viability of tumor cells as % of untreated control.Treatment with pemetrexed (ALIMTA®) showed enhanced tumor growthinhibition in the combination with ABR-217620.

TABLE 5 Pemetrexed (ALIMTA ®) enhances tumor growth inhibition in thecombination with ABR-217620. Effect on human tumor cell (Colo 205)viability, SADCC, 4 h Chemo, 48 h Percent of control Vehicle Vehicle 100ABR-217620, Vehicle 79 1 pM Vehicle Pemetrexed, 78 40 ng/ml ABR-217620,Pemetrexed, 60 1 pM 40 ng/ml

The above results showed that the combination of the chemotherapeuticagent pemetrexed (ALIMTA®) with TTS enhanced the tumor growth inhibitioninduced by TTS.

Example 4 TTS-Chemotherapeutic Agent (e.g., Docetaxel) CombinationTherapy In Vitro Anti-Tumor

The effect of superantigen (TTS) treatment (5T4Fab-SEA/E-120) incombination with cytostatics on tumor cell killing was assessed invitro.

Cell Lines and Media

The human cancer cell line Colo 205 was cultured in RPMI 1640+10% FBS(R10 medium). The cells were prepared by detachment with celldissociation solution, washed with PBS and resuspended in R10 medium.Peripheral blood mononuclear cells (PBMC) were obtained from blooddonors. The PBMC were isolated by density centrifugation onFicoll-Paque. The cells were then cultured in R10 medium and stimulatedwith TTS to obtain effector T-cell lines.

Tumor Cell Killing by Superantigen Antibody Dependent Cell-MediatedCytotoxicity; SADCC

To analyze anti-tumor activity of combinations of TTS (ABR-217620) anddocetaxel (TAXOTERE®) in vitro a chromium-release assay was applied. Thetarget cells in the cytotoxicity assay, human Colo 205 tumor cells, werefirst pre-treated with various concentrations of docetaxel for 16 hours,washed in medium and then labeled with (Na)₂ ⁵¹CrO₄. The anti-tumorcytotoxicity (Superantigen Antibody Dependent Cell-mediatedCytotoxicity; SADCC) was then measured in the presence TTS and humanactivated effector T cells in a standard chromium release assay using96-well plates. The percentage specific lysis was calculated accordingto the formula:

${\%\mspace{14mu}{specific}\mspace{14mu}{lysis}} = {\frac{{{experimental}\mspace{14mu}{release}} - {{spontaneous}\mspace{14mu}{release}}}{{{total}\mspace{14mu}{release}} - {{spontaneous}\mspace{14mu}{release}}} \times 100}$Results

Pre-treatment of the human Colo 205 tumor cells with docetaxel(TAXOTERE®) induced enhanced sensitivity for activated T cells andABR-217620, Superantigen Antibody Dependent Cell-mediated Cytotoxicity;SADCC. FIG. 4 depicts killing of tumor cells as % specific lysis withvarious concentrations of ABR-217620 and activated T cells.Pre-treatment with docetaxel (TAXOTERE®) showed enhanced tumor cellkilling as compared to control tumor cells.

The results showed that the combination of the chemotherapeutic agentdocetaxel (TAXOTERE®) as sequential pre-treatment together with TTSenhanced the tumor cell killing induced by TTS.

Example 5 TTS-Chemotherapeutic (e.g., Gemcitabine) Combination TherapyIn Vivo Examples of Systemic Immune Activation in Mice

The systemic immune response was evaluated in C57B1/6 mice followingtreatment with C215Fab-SEA in combination with the chemotherapeuticagent gemcitabine.

Animals

Female C57B1/6 mice were used. The mice were routinely used at the ageof 8 to 12 weeks and received food and water ad libitum.

Cell Lines and Media

The murine B-cell lymphoma cell line A20 (ATCC, TIB-208) was maintainedin RPMI 1640 with ultraglutamine supplemented with 10% FCS, 1 mM sodiumpyruvate, 50 μM β-mercaptoethanol and 0.1 mg/ml gentamicin.

Treatment of Mice

C215Fab-SEA treatment was given as daily i.v. injections (10 μg/mouse,in 0.2 ml PBS containing 1% mouse serum) for 3 consecutive days. Forcombination studies, four doses of gemcitabine were injected i.p. withthree days interval before C215Fab-SEA therapy. Three mice were includedin each experimental group. 48 hours after the last C215Fab-SEAinjection mice were sacrificed and the spleens were removed for analysisof cell dynamics and cytotoxic activity.

Flow Cytometry

Single cell suspensions of splenocytes from mice injected with indicatedsubstances were prepared. Erythrocytes were removed by hypotonic shockusing Gey's solution. The remaining lymphocytes were stained for cellsurface antigen expression for 30 min on ice after blocking ofFc-receptors by CD32/CD16 antibodies. Where biotinylated mAbs were used,cells were stained with streptavidin-tricolor. Cells were washed twicein PBS+1% BSA after each staining step. The samples were analysed in aFACSort flow cytometer.

Cytotoxicity Assays

Mice treated with indicated substances were sacrificed 48 h after thelast injection and the spleens were removed. Cytotoxicity of bulksplenocytes was measured against SEA coated (1 μg/ml) A20 cells using astandard ⁵¹Cr release assay (Dohlsten et al., 1990; Hedlund et al,1993). The effector to target cell ratio was 100:1. Calculations werecarried out according to the formula:

${\%\mspace{14mu}{specific}\mspace{14mu}{lysis}} = {\frac{{{experimental}\mspace{14mu}{release}} - {{spontaneous}\mspace{14mu}{release}}}{{{total}\mspace{14mu}{release}} - {{spontaneous}\mspace{14mu}{release}}} \times 100}$ResultsPre-Treatment with Chemotherapeutic (Gemcitabine) does not NegativelyInterfere with Sea Induced T-Cell Activation

Standard treatment of tumor free C57B1/6 mice with gemcitabine (fourdoses with three days interval) at various doses was followed by 3 dailyinjections of C215Fab-SEA starting 6 days after the last gemcitabineinjection. Splenocytes were prepared 48 hours after the last injectionand superantigen dependent cellular cytotoxicity (SDCC) was measuredagainst SEA coated A20 cells in a standard ⁵¹Cr release assay.Substantial cytotoxic activity was detected in all Fab-SEA treatedgroups irrespective of prior gemcitabine therapy (FIG. 5). Flowcytometric analysis showed that SEA induced CD8-V_(β)3 T-cell expansionincreased by gemcitabine treatment (FIG. 6C). No significant T-cellactivation or SDCC was found in mice treated with gemcitabine alone atany dose (FIG. 6).

To investigate the effect of prolonged gemcitabine treatment on SEAinduced immune activation, mice were given 7 injections of gemcitabinewith 3 days interval, followed by C215Fab-SEA treatment starting 6 daysafter the last gemcitabine injection. As shown in FIGS. 7 and 8, thisprolonged treatment schedule did not significantly alter theresponsiveness to SEA.

Next, the impact of the length of the treatment free period betweengemcitabine therapy and TTS treatment on SEA induced T-cell activationwas evaluated. Following standard therapy with gemcitabine at 2.4mg/mouse, three daily injections of C215Fab-SEA were given, starting 1,3, or 6 days after the last gemcitabine administration. Spleens wereremoved 48 hours post treatment and splenocytes were analysed for T-cellactivation and effector functions (SDCC). Interestingly, shortening ofthe rest period between treatments resulted in potentiation of V_(β)3specific T-cell expansion characteristic of a SEA induced immuneresponse (FIG. 9). This was accompanied by enhanced CTL activity ofsplenocytes against SEA coated A20 cells (FIG. 10).

These results indicated that pre-treatment with gemcitabine did notinterfere with TTS therapy and may even act to improve SEA inducedeffector functions.

Example 6 TTS-Chemotherapeutic (e.g., Docetaxel) Combination Therapy InVivo, Systemic Immune Activation in Mice

The systemic immune response was evaluated in C57B1/6 mice followingtreatment with C215Fab-SEA in combination with the cytostatic agentdocetaxel.

Animals

Female C57B1/6 mice were used. The mice were routinely used at the ageof 8 to 12 weeks and received food and water ad libitum.

Cell Lines and Media

The murine B-cell lymphoma cell line A20 (ATCC, TIB-208) was maintainedin RPMI 1640 with ultraglutamine supplemented with 10% FCS, 1 mM sodiumpyruvate, 50 μM β-mercaptoethanol and 0.1 mg/ml gentamicin.

Treatment of Mice

C215Fab-SEA treatment was given as daily i.v. injections (10 μg/mouse,in 0.2 ml PBS containing 1% mouse serum) for 3 consecutive days. Incombination studies, one i.p. injection of docetaxel was given (in 0.2ml buffer solution containing polysorbate (0.1-10.9%), ethanol(0.1-10.1%) and NaCl (0.45-0.9%)) either before or simultaneously withC215Fab-SEA therapy. Three mice were included in each experimentalgroup. 48 hours after the last C215Fab-SEA injection, mice weresacrificed and the spleens were removed for analysis of T-cell dynamicsand cytotoxic activity.

Flow Cytometry

Single cell suspensions of splenocytes from mice injected with indicatedsubstances were prepared. Erythrocytes were removed by hypotonic shockusing Gey's solution. The remaining lymphocytes were stained for cellsurface antigen expression for 30 min on ice after blocking ofFc-receptors by CD32/CD16 antibodies. Where biotinylated mAbs were used,cells were stained with streptavidin-tricolor. Cells were washed twicein PBS+1% BSA after each staining step. The samples were analysed in aFACSort flow cytometer.

Cytotoxicity Assays

Mice treated with indicated substances were sacrificed 48 h after thelast injection and the spleens were removed. Cytotoxicity of bulksplenocytes was measured against SEA coated (1 μg/ml) A20 cells using astandard ⁵¹Cr release assay (Dohlsten et al., 1990; Hedlund et al,1993). The effector to target cell ratio was 100:1. Calculations werecarried out according to the formula:

${\%\mspace{14mu}{specific}\mspace{14mu}{lysis}} = {\frac{{{experimental}\mspace{14mu}{release}} - {{spontaneous}\mspace{14mu}{release}}}{{{total}\mspace{14mu}{release}} - {{spontaneous}\mspace{14mu}{release}}} \times 100}$ResultsPre-Treatment with Chemotherapeutic (Docetaxel) does not NegativelyInterfere with SEA-Induced T-Cell Activation

Treatment of C57B1/6 mice with various doses of docetaxel on day 1 wasfollowed by 3 daily injections of C215Fab-SEA starting at day 2.Splenocytes were prepared 48 hours after the last injection andsuperantigen dependent cellular cytotoxicity (SDCC) was measured againstSEA coated A20 cells in a standard ⁵¹Cr release assay. Substantialcytotoxic activity (FIG. 11) and V_(β)3 specific T-cell expansion (FIG.12) were detected in all Fab-SEA treated groups irrespective of priordocetaxel therapy. Thus, cytostatic treatment before TTS therapyapparently did not interfere with SEA mediated immune activation.Rather, administration of therapeutic doses of docetaxel (1 mg/animal)shortly before TTS treatment resulted in potentiation of SEA inducedT-cell activation as measured by SDCC (FIG. 11). No significant T-cellactivation or SDCC was recorded in mice treated with docetaxel alone atany dose. The highest docetaxel dose used in this experiment (1 mg) wassufficient to induce significant anti-tumor effects in tumor bearinganimals when administered alone and to augment of TTS therapy the effectwhen combined with C215Fab-SEA.

Example 7 TTS-Chemotherapeutic (e.g., Gemcitabine) Combination TherapyIn Vivo—Synergistic Effects in Mice

C215Fab-SEA (ABR-214720) in combination with gemcitabine wasinvestigated with regard to tumor therapy efficacy in C57B1/6 mice. Micewere inoculated i.v. with B16C215 melanoma cells to induce lung tumorsin the therapy studies. Gemcitabine was administered after the firstC215Fab-SEA therapy cycle.

Cells

The murine B16 melanoma cell line (obtained from ATCC) transfected withthe human antigen C215 was cultured in cell growth medium. The cell linewas prepared by detachment of cells grown in vitro with celldissociation solution, resuspended in PBS, counted and diluted in PBSwith 1% C57B1/6 mouse serum.

Animals

Female C57B1/6 mice were used. The mice were routinely used at the ageof 8 to 12 weeks and received food and water ad libitum.

Tumor Therapy In C57B1/6 Mice: Number of Lung Tumors

Groups of eight C57B1/6 mice were inoculated i.v. with 1.75×10⁵ B16melanoma cells transfected with the C215 human tumor antigen (Dohlsten,M. et al., 1994) into the tail vein, in 0.2 ml vehicle, to induce lungtumors. The chemotherapeutic agent was injected i.p. on various days in0.2 ml vehicle. For each treatment cycle ABR-214720 was injected i.v.with four daily injections in 0.2 ml vehicle. Three to 6 weeks later themice were sacrificed and the lungs were removed. After fixation inBouins solution for at least 24 h, the numbers of lung tumors werecounted.

Treatment Regimens

Ten μs/mouse of ABR-214720 was given days 3-6 and 18-21 and 2.4 mg/mouseof gemcitabine was given days 2, 5, 8 and 11 (only mono-therapy) or days8, 11, 14 and 17 as mono-therapies or in combination, respectively.

Results

As shown in Table 6, administration of 2.4 mg gemcitabine per animal ondays 2, 5, 8 and 11 was active as a single agent in this model,demonstrating the in vivo sensitivity of B16 tumor cells to gemcitabine.

TABLE 6 Therapy of tumor metastases: Number of lung tumors per animalafter treatment with gemcitabine. Mean number of lung metastasesTreatment day 21 Control* 147 Gemcitabine 2.4 mg/mouse 49.5 *non-treatedtumor inoculated animals

As illustrated in Table 7, combination therapy was superior andsynergistic as compared to treatment with single treatment gemcitabineor TTS, in reducing the number of lung metastases. Single treatment TTSresulted in significant anti-tumor effect. Gemcitabine was administeredfrom day 8 after tumor inoculation at a dosage shown to be tumor growthinhibitory in mice with less pronounced disease, but did not exert anyanti-tumor effect per se on this rapidly growing tumor at this stage.TTS and gemcitabine doses were selected to exert maximal anti-tumoreffects.

TABLE 7 Therapy of tumor metastases: Number of lung tumors per animalafter treatment with the drugs alone or in combination Mean number oflung Mean number of lung Treatment metastases Day 28 metastases Day 40Control* 117 All animals dead TTS 23 21 Gemcitabine 141 All animals deadTTS + gemcitabine 11 10 *non-treated tumor inoculated animals

Thus, the combination of TTS with chemotherapeutic agents for examplegemcitabine resulted in synergistic anti-tumor effects when used insequence and starting with TTS.

Example 8 TTS-Chemotherapeutic (e.g., Docetaxel) Combination Therapy InVivo—Synergistic Effects in Mice

C215Fab-SEA (ABR-214720) in combination with docetaxel was investigatedwith regard to tumor therapy efficacy in C57B1/6 mice. Mice wereinoculated i.v. with B16C215 melanoma cells to induce lung tumors in thetherapy studies. Docetaxel was administered before and after as well asduring the C215Fab-SEA therapy cycle.

Cells

The murine B16 melanoma cell line (obtained from ATCC) transfected withthe human antigen C215 was cultured in cell growth medium. The cell linewas prepared by detachment of cells grown in vitro with celldissociation solution, resuspended in PBS, counted and diluted in PBSwith 1% C57B1/6 mouse serum.

Animals

Female C57B1/6 mice were used. The mice were routinely used at the ageof 8 to 12 weeks and received food and water ad libitum.

Tumor Therapy in C57B1/6 Mice: Number of Lung Tumors

Groups of eight C57B1/6 mice were inoculated i.v. with 1.75×10⁵ B16melanoma cells transfected with the C215 human tumor antigen (Dohlsten,M. et al., 1994) into the tail vein, in 0.2 ml vehicle, to induce lungtumors. The chemotherapeutic agent was injected i.p. on various days in0.2 ml vehicle. For each treatment cycle ABR-214720 was injected i.v.with four daily injections in 0.2 ml vehicle. Three to 6 weeks later themice were sacrificed and the lungs were removed. After fixation inBouins solution for at least 24 h, the numbers of lung tumors werecounted.

Treatment Regimens

Ten μg/mouse of ABR-214720 was given days 3-6 and 1.0 mg/mouse ofdocetaxel was given days 2 and 9 or day 5 as mono-therapies or incombination, respectively.

Results

As can be seen from Table 8, the combination therapy was superior andsynergistic as compared to treatment with docetaxel (days 2 and 9) orTTS only, in reducing the number of lung metastases. Treatment with TTSor docetaxel alone resulted in significant anti-tumor effects. However,treatment with TTS in combination with docetaxel exerted maximalanti-tumor effects.

TABLE 8 Therapy of tumor metastases: Number of lung tumors per animalafter treatment with the drugs alone or in combination. Treatment Meannumber of lung metastases day 21 Control* 137 TTS 19 Docetaxel 48 TTS +Docetaxel 1 *non-treated tumor inoculated animals

As can be seen from Table 9, the combination therapy was superior andsynergistic as compared to treatment with docetaxel (day 5) or TTS only,in reducing the number of lung metastases. Treatment with TTS aloneresulted in significant anti-tumor effects. However, treatment with TTSin combination with docetaxel exerted maximal anti-tumor effects.

TABLE 9 Therapy of tumor metastases: Number of lung tumors per animalafter treatment with the drugs alone or in combination. Treatment Meannumber of lung metastases day 21 Control* 73 TTS 14 Docetaxel 93 TTS +Docetaxel 6 *non-treated tumor inoculated animals

Thus, the combination of TTS with chemotherapeutic agents for exampledocetaxel resulted in synergistic anti-tumor effects when used insequence with TTS as well as integrated on the third day of the TTScycle.

Example 9 TTS-Chemotherapeutic (e.g., Docetaxel) Combination Therapy InVivo—Synergistic Effects in Mice

C215Fab-SEA (ABR-214720) in combination with docetaxel was investigatedwith regard to tumor therapy efficacy in C57B1/6 mice as measured bysurvival. Mice were inoculated i.v. with B16C215 melanoma cells toinduce lung tumors in the therapy studies. Docetaxel was administeredbefore or before and after the C215Fab-SEA therapy cycle.

Cells

The murine B16 melanoma cell line (obtained from ATCC) transfected withthe human antigen C215 was cultured in cell growth medium. The cell linewas prepared by detachment of cells grown in vitro with celldissociation solution, resuspended in PBS, counted and diluted in PBSwith 1% C57B1/6 mouse serum.

Animals

Female C57B1/6 mice were used. The mice were routinely used at the ageof 8 to 12 weeks and received food and water ad libitum.

Tumor Therapy in C57B1/6 Mice: Survival

Groups of 8 to 20 C57B1/6 mice were inoculated i.v. with 1.75×10⁵ B16melanoma cells transfected with the C215 human tumor antigen (Dohlsten,M., et al. 1994) into the tail vein, in 0.2 ml vehicle, to induce lungtumors. The chemotherapeutic agent was injected i.p. on various days in0.2 ml vehicle. For each treatment cycle ABR-214720 was injected i.v.with four daily injections in 0.2 ml vehicle. When the mice showed signsof morbidity or on day 90 or 120, they were sacrificed and an autopsywas carried out.

Treatment Regimens

Ten μg/mouse of ABR-214720 was given days 3-6 and 1.0 mg/mouse ofdocetaxel was given day 2 or days 2 and 9 as mono-therapies or incombination, respectively.

Results

As illustrated in Table 10, combination therapy was superior andsynergistic as compared to treatment with single treatment docetaxel orTTS, in increasing longevity. Single treatment TTS or docetaxel resultedin significant anti-tumor effects. TTS and docetaxel doses were selectedto exert maximal anti-tumor effects.

TABLE 10 Therapy of tumor metastases: Median survival time of micewithlung tumor metastasis after treatment with the drugs alone or incombination. Treatment Median survival time (Days) Control* 33 TTS 41.5Docetaxel (Day 2) 38 Docetaxel (Days 2 and 9) 37.5 TTS + Docetaxel (Day2) 66 TTS + Docetaxel (Days 2 and 9) >90 *non-treated tumor inoculatedanimals

Thus, the combination of TTS with chemotherapeutic agents for exampledocetaxel resulted in synergistic anti-tumor effects when used insequence and starting with the chemotherapeutic agent.

Example 10 TTS-Chemotherapeutic (e.g., Docetaxel) Combination Therapy InVivo—Synergistic Effects in Mice

C215Fab-SEA (ABR-214720) in combination with docetaxel was investigatedwith regard to tumor therapy efficacy in C57B1/6 mice as measured bysurvival. Mice were inoculated i.v. with B16C215 melanoma cells toinduce lung tumors in the therapy studies. Docetaxel was administeredafter the C215Fab-SEA therapy cycles.

Cells

The murine B16 melanoma cell line (obtained from ATCC) transfected withthe human antigen C215 was cultured in cell growth medium. The cell linewas prepared by detachment of cells grown in vitro with celldissociation solution, resuspended in PBS, counted and diluted in PBSwith 1% C57B1/6 mouse serum.

Animals

Female C57B1/6 mice were used. The mice were routinely used at the ageof 8 to 12 weeks and received food and water ad libitum.

Tumor Therapy in C57B1/6 Mice: Survival

Groups of 8 to 20 C57B1/6 mice were inoculated i.v. with 1.75×10⁵ B16melanoma cells transfected with the C215 human tumor antigen (Dohlsten,M., et al. 1994) into the tail vein, in 0.2 ml vehicle, to induce lungtumors. The chemotherapeutic agent was injected i.p. on various days in0.2 ml vehicle. For each treatment cycle ABR-214720 was injected i.v.with four daily injections in 0.2 ml vehicle. When the mice showed signsof morbidity or on day 90 or 120, they were sacrificed and an autopsywas carried out.

Treatment Regimens

Ten μg/mouse of ABR-214720 was given days 3-6 and 31-34 and 2.0 mg/mouseof docetaxel was given days 8, 22, 36 and 50 as mono-therapies or incombination, respectively.

Results

As illustrated in Table 11, combination therapy was superior andsynergistic as compared to treatment with single treatment docetaxel orTTS, in increasing longevity. Single treatment TTS or docetaxel resultedin significant anti-tumor effects. TTS and docetaxel doses were selectedto exert maximal anti-tumor effects.

TABLE 11 Therapy of tumor metastases: Median survival time of mice withlung tumor metastasis after treatment with the drugs alone or incombination. Treatment Median survival time (Days) Control* 31.5 TTS 48Docetaxel 50 TTS + Docetaxel 75 *non-treated tumor inoculated animals

Thus, the combination of TTS with chemotherapeutic agents for exampledocetaxel resulted in synergistic anti-tumor effects when used insequence and starting with TTS.

Example 11 TTS-Chemotherapeutic (e.g., Docetaxel) Combination Therapy InVivo—Synergistic Effects in Mice

C215Fab-SEA (ABR-214720) in combination with docetaxel was investigatedwith regard to tumor therapy efficacy in C57B1/6 mice as measured bysurvival. Mice were inoculated i.v. with B16C215 melanoma cells toinduce lung tumors in the therapy studies. Docetaxel was administeredthe day immediately after the last day of the C215Fab-SEA therapycycles.

Cells

The murine B16 melanoma cell line (obtained from ATCC) transfected withthe human antigen C215 was cultured in cell growth medium. The cell linewas prepared by detachment of cells grown in vitro with celldissociation solution, resuspended in PBS, counted and diluted in PBSwith 1% C57B1/6 mouse serum.

Animals

Female C57B1/6 mice were used. The mice were routinely used at the ageof 8 to 12 weeks and received food and water ad libitum.

Tumor Therapy in C57B1/6 Mice: Survival

Groups of 8 to 20 C57B1/6 mice were inoculated i.v. with 1.75×10⁵ B16melanoma cells transfected with the C215 human tumor antigen (Dohlsten,M., et al. 1994) into the tail vein, in 0.2 ml vehicle, to induce lungtumors. The chemotherapeutic agent was injected i.p. on various days in0.2 ml vehicle. For each treatment cycle ABR-214720 was injected i.v.with four daily injections in 0.2 ml vehicle. When the mice showed signsof morbidity or on day 90 or 120, they were sacrificed and an autopsywas carried out.

Treatment Regimens

Ten μg/mouse of ABR-214720 was given days 3-6, 17-20, 45-48 and 59-62and 2.0 mg/mouse of docetaxel was given days 7, 21, 35, 49, 63 and 77 asmono-therapies or in combination, respectively.

Results

As illustrated in Table 12, combination therapy was superior andsynergistic as compared to treatment with single treatment docetaxel orTTS, in increasing longevity. Single treatment TTS or docetaxel resultedin significant anti-tumor effects. TTS and docetaxel doses were selectedto exert maximal anti-tumor effects.

TABLE 12 Therapy of tumor metastases: Median survival time of mice withlung tumor metastasis after treatment with the drugs alone or incombination. Treatment Median survival time (Days) Control* 35 TTS 75Docetaxel 61 TTS + Docetaxel >120 *non-treated tumor inoculated animals

Thus, the combination of TTS with chemotherapeutic agents for exampledocetaxel resulted in synergistic anti-tumor effects when used insequence and starting with TTS and giving the chemotherapeutic agent theday immediately after the last day of the TTS treatment cycles.

Example 12 TTS-Chemotherapeutic (e.g., Gemcitabine) Combination TherapyIn Vivo—Humanized SCID Mice Model

The effect of TTS treatment (5T4Fab-SEA/E-120, ABR-217620) incombination with the chemotherapeutic agent gemcitabine on tumor growthwas assessed in an experimental human tumor model. Severe CombinedImmunodeficient (SCID) mice were transplanted with human lymphocytes andhuman tumor cells growing intraperitoneally as solid tumors. ABR-217620is a TTS developed for human use and therefore tested in the humanizedSCID model. The anti-tumor TTS effects are dependent of activated humanlymphocytes.

Cells

The human tumor cell line Colo 205 (obtained from ATCC) was cultured incell growth medium. The cell line was prepared by detachment of cellsgrown in vitro with cell dissociation solution, resuspended in PBS,counted and diluted in PBS with 1% SCID mouse serum. Peripheral bloodmononuclear cells (PBMC) were obtained from blood donors at theUniversity Hospital of Lund. The PBMC were isolated by densitycentrifugation on Ficoll-Paque. The cells were then cultured in R10medium and stimulated with TTS to obtain activated effector Tlymphocytes. The T lymphocytes were prepared by detachment of cellsgrown in vitro, resuspended in PBS, counted and diluted in PBS with 1%SCID mouse serum.

Animals

Female SCID mice were used. The mice were routinely used at the age of 8to 12 weeks and received sterile pelleted rodent diet and sterile waterad libitum.

Tumor Therapy in SCID Mice: Intraperitoneal Tumor Mass

Seven to 8 SCID mice per treatment group were injected intraperitoneallywith human tumor cells in 0.2 ml vehicle. The mice received anintraperitoneal injection with activated lymphocytes in 0.2 ml vehicle,and additionally they received 4-5 daily intravenous injections with TTStest substance in 0.2 ml vehicle, the first injection at the same timeas the activated lymphocytes were given. The TTS treatment was combinedwith intraperitoneal (i.p.) or intravenous (i.v.) injections of thechemotherapeutic agent. After 3-10 weeks the mice were sacrifised byneck dislocation and the tumor mass was determined.

Treatment Regimens

Fifty μg/mouse of ABR-217620 was given days 2-6 after tumor injectionand 2.0 mg/mouse of docetaxel was given i.v. days 3, 5 and 8 asmono-therapies or in combination, respectively.

Results

As can be seen from Table 13, the combination therapy was superior andsynergistic as compared to treatment with gemcitabine or TTS only, inreducing the tumor mass. Treatment with TTS alone resulted insignificant anti-tumor effects. However, treatment with TTS incombination with gemcitabine exerted maximal anti-tumor effects.

TABLE 13 Therapy of human tumors: Tumor mass per animal after treatmentwith the drugs alone or in combination. Treatment Mean/Median Tumor Mass(mg) Control* 403/362 TTS 133/79  Gemcitabine 303/300 TTS + Gemcitabine60/43 *non-treated tumor inoculated animals

Thus, the combination of TTS with chemotherapeutic agents for examplegemcitabine resulted in synergistic anti-tumor effects when usedintegrated, starting closely before the TTS cycle.

Example 13 TTS-Chemotherapeutic (e.g., Docetaxel) Combination Therapy InVivo—Humanized SCID Mice Model

The effect of TTS treatment (5T4Fab-SEA/E-120, ABR-217620) incombination with the chemotherapeutic agent docetaxel on tumor growthwas assessed in an experimental human tumor model. Severe CombinedImmunodeficient (SCID) mice were transplanted with human lymphocytes andhuman tumor cells growing intraperitoneally as solid tumors. ABR-217620is a TTS developed for human use and therefore tested in the humanizedSCID model. The anti-tumor TTS effects are dependent of activated humanlymphocytes.

Cells

The human tumor cell line Colo 205 (obtained from ATCC) was cultured incell growth medium. The cell line was prepared by detachment of cellsgrown in vitro with cell dissociation solution, resuspended in PBS,counted and diluted in PBS with 1% SCID mouse serum. Peripheral bloodmononuclear cells (PBMC) were obtained from blood donors at theUniversity Hospital of Lund. The PBMC were isolated by densitycentrifugation on Ficoll-Paque. The cells were then cultured in R10medium and stimulated with TTS to obtain activated effector Tlymphocytes. The T lymphocytes were prepared by detachment of cellsgrown in vitro, resuspended in PBS, counted and diluted in PBS with 1%SCID mouse serum.

Animals

Female SCID mice were used. The mice were routinely used at the age of 8to 12 weeks and received sterile pelleted rodent diet and sterile waterad libitum.

Tumor Therapy in SCID Mice: Intraperitoneal Tumor Mass

Seven to 8 SCID mice per treatment group were injected intraperitoneallywith human tumor cells in 0.2 ml vehicle. The mice received anintraperitoneal injection with activated lymphocytes in 0.2 ml vehicle,and additionally they received 4-5 daily intravenous injections with TTStest substance in 0.2 ml vehicle, the first injection at the same timeas the activated lymphocytes were given. The TTS treatment was combinedwith intraperitoneal (i.p.) or intravenous (i.v.) injections of thechemotherapeutic agent. After 3-10 weeks the mice were sacrifised byneck dislocation and the tumor mass was determined.

Treatment Regimens

Fifty μg/mouse of ABR-217620 was given days 6-10 after tumor injectionand 0.2 mg/mouse of docetaxel was given i.p. day 4 as mono-therapies orin combination, respectively.

Results

As can be seen from Table 14, the combination therapy was superior andsynergistic as compared to treatment with docetaxel or TTS only, inreducing the tumor mass. Treatment with docetaxel alone resulted insignificant anti-tumor effects. However, treatment with TTS incombination with gemcitabine exerted maximal anti-tumor effects.

TABLE 14 Therapy of human tumors: Tumor mass per animal after treatmentwith the drugs alone or in combination. Treatment Mean/Median Tumor Mass(mg) Control* 321/282 TTS 315/316 Docetaxel 176/103 TTS + Docetaxel52/46 *non-treated tumor inoculated animals

Thus, the combination of TTS with chemotherapeutic agents for exampledocetaxel resulted in synergistic anti-tumor effects when thechemotherapeutic agent is used closely before the TTS cycle.

Example 14 TTS-Chemotherapeutic (e.g., Docetaxel) Combination Therapy InVivo—Humanized SCID Mice Model

The effect of TTS treatment (5T4Fab-SEA/E-120, ABR-217620) incombination with the chemotherapeutic agent docetaxel on tumor growthwas assessed in an experimental human tumor model. Severe CombinedImmunodeficient (SCID) mice were transplanted with human lymphocytes andhuman tumor cells growing intraperitoneally as solid tumors. ABR-217620is a TTS developed for human use and therefore tested in the humanizedSCID model. The anti-tumor TTS effects are dependent of activated humanlymphocytes.

Cells

The human tumor cell line Calu-1 (obtained from ATCC) was cultured incell growth medium. The cell line was prepared by detachment of cellsgrown in vitro with cell dissociation solution, resuspended in PBS,counted and diluted in PBS with 1% SCID mouse serum. Peripheral bloodmononuclear cells (PBMC) were obtained from blood donors at theUniversity Hospital of Lund. The PBMC were isolated by densitycentrifugation on Ficoll-Paque. The cells were then cultured in R10medium and stimulated with TTS to obtain activated effector Tlymphocytes. The T lymphocytes were prepared by detachment of cellsgrown in vitro, resuspended in PBS, counted and diluted in PBS with 1%SCID mouse serum.

Animals

Female SCID mice were used. The mice were routinely used at the age of 8to 12 weeks and received sterile pelleted rodent diet and sterile waterad libitum.

Tumor Therapy in SCID Mice: Intraperitoneal Tumor Mass

Seven to 8 SCID mice per treatment group were injected intraperitoneallywith human tumor cells in 0.2 ml vehicle. The mice received anintraperitoneal injection with activated lymphocytes in 0.2 ml vehicle,and additionally they received 4-5 daily intravenous injections with TTStest substance in 0.2 ml vehicle, the first injection at the same timeas the activated lymphocytes were given. The TTS treatment was combinedwith intraperitoneal (i.p.) or intravenous (i.v.) injections of thechemotherapeutic agent. After 3-10 weeks the mice were sacrifised byneck dislocation and the tumor mass was determined.

Treatment Regimens

Fifty μg/mouse of ABR-217620 was given days 2-6 after tumor injectionand 0.2 mg/mouse of docetaxel was given i.p. day 7 as mono-therapies orin combination, respectively.

Results and Discussion

As can be seen from Table 15, the combination therapy was superior andsynergistic as compared to treatment with docetaxel or TTS only, inreducing the tumor mass. Treatment with docetaxel alone resulted insignificant anti-tumor effects. However, treatment with TTS incombination with gemcitabine exerted maximal anti-tumor effects.

TABLE 15 Therapy of human tumors: Tumor mass per animal after treatmentwith the drugs alone or in combination. Treatment Mean/Median Tumor Mass(mg) Control* 156/39  TTS 221/79  Docetaxel 31/29 TTS + Docetaxel 9/5*non-treated tumor inoculated animals

Thus, the combination of TTS with chemotherapeutic agents for exampledocetaxel resulted in synergistic anti-tumor effects when thechemotherapeutic agent is used closely after the TTS cycle.

Example 15 TTS-Chemotherapeutic Combination Therapy In Vivo—Inhibitionof Anti-TTS Antibody Formation

It is of vital importance that high titer antibodies do not develop andneutralize the TTS drug. C215Fab-SEA (ABR-214720) in combination withgemcitabine was investigated with regard to modulation of the anti-SEAantibody response in C57B1/6 mice. Primary anti-SEA antibody responsewas induced by immunization with SEA. ABR-214720 treatment was given as4 daily i.v. injections. For combination studies, gemcitabine wasinjected four times at three days intervals starting either beforecompleting TTS therapy or shortly after the last ABR-214720 injection.

Animals

Female C57B1/6 mice were used. The mice were routinely used at the ageof 8 to 12 weeks and received food and water ad libitum.

Determination of Mouse Anti-SEA Antibodies

The concentration of anti-SEA antibodies in the mouse plasma samples wasdetermined by ELISA technique. The assay was performed with reagentaddition, incubation and washing in a sequential manner. In the firststep the microtiter plate wells were coated with 1 μg/mL (200 ng/well)of recombinant SEA in 50 mM NaHCO₃, pH 9.6. The wells were blocked with1% (w/v) bovine serum albumin (BSA) in 10 mM PBS, pH 7.4, 0.05% (w/v)Tween 20 (PBST). Samples and standards of affinity purified mouseanti-SEA antibodies were added, diluted in 1% BSA in PBST. A goatanti-mouse IgG antibody, was used as secondary antibody. Next, abiotinylated rabbit anti-goat IgG antibody was added. Thereafter,streptavidin conjugated with horseradish peroxidase was allowed to bindto the biotin groups of the tertiary antibody. The final step wasaddition of an enzyme substrate. The enzyme reaction was stopped by 1NH₂SO₄ and then the absorbance was monitored at 450 nm, with 650 nm as areference wavelength in an ELISA spectrophotometer. A four-parameterfunction was adjusted to the obtained concentration/absorbance values ofthe standards and the unknown concentration of anti-SEA antibodies insamples determined from standard curve. The measuring range wasestimated to 0.40-50 ng/mL. The sample dilution was at least 1:100, i.e.the limit of quantification (LOQ) in plasma was 40 ng/mL.

Induction of Anti-SEA Antibodies and Treatment with ChemotherapeuticAgents in C57B1/6 Mice

For induction of a primary anti-SEA response, mice were given 4intravenous injections of unconjugated SEA (3 μg/mouse/injection, in 0.2ml PBS containing 1% mouse serum) on days 1, 5, 9 and 13. Starting onday 29 after priming, ABR-214720 treatment was given as daily i.v.injections (10 μg/mouse/injection, in 0.2 ml PBS containing 1% mouseserum) on 4 consecutive days. Gemcitabine was injected (2.4.mg/mouse/injection i.p. in 0.2 ml 0.15M NaCl) four times at three daysinterval, starting at indicated time points during or after completionof ABR-214720 treatment. At indicated time points blood samples weredrained by vena saphena or vena cava for preparation of plasma. Eightmice were included in each experimental group.

Results

As illustrated in Table 16, combination therapy was superior as comparedto treatment with single treatment TTS, in maintaining a low anti-SEAantibody titer. Single treatment TTS resulted in significant anti-SEAantibody titers. Gemcitabine was administered beginning during or afterthe TTS treatment cycle. It was clearly shown that the combinationtreatment starting both during as well as after the TTS cycle resultedin much lower secondary response anti-SEA antibody titers as compared tothe TTS treatment alone (groups 4-7 as compared to group 3).

TABLE 16 Inhibition of anti-SEA antibodies by combination withchemotherapy. Anti-SEA day 28 Anti-SEA day 46 Ratio Group SEA (i.v.)C215Fab-SEA (i.v.) Gemcitabine (i.p) (ng/ml) (ng/ml) Secondary/primaryNo Days 1, 5, 9, 13 Days 29, 30, 31, 32 Start day Primary responseSecondary response response 1 Vehicle Vehicle Vehicle, d 34 59 97 1.6 2SEA Vehicle Vehicle, d 34 1073 337 0.3 3 SEA C215Fab-SEA Vehicle, d 34333 6648 20.0 4 SEA C215Fab-SEA Gemcitabine d 30 749 166 0.2 5 SEAC215Fab-SEA Gemcitabine d 32 353 197 0.5 6 SEA C215Fab-SEA Gemcitabine d33 797 747 0.9 7 SEA C215Fab-SEA Gemcitabine d 34 1139 4084 3.6

Starting gemcitabine treatment before integrated TTS injections as wellas after TTS injections resulted in a much lower secondary responseanti-SEA antibody titers as compared to the TTS treatment alone. Thus,the combination of TTS with chemotherapeutic agents for examplegemcitabine resulted in lowered levels of anti-Superantigen (anti-Sag)antibodies after TTS treatment cycles. Additional TTS treatment cyclescan therefore be given without interference from neutralizing high titeranti-Sag antibodies.

Example 16 TTS-Chemotherapeutic Combination Therapy In Vivo—Inhibitionof Anti-TTS Antibody Formation

It is of vital importance that high titer antibodies do not develop andneutralize the TTS drug. C215Fab-SEA (ABR-214720) in combination withdocetaxel was investigated with regard to modulation of the anti-SEAantibody response in C57B1/6 mice. Primary anti-SEA antibody responsewas induced by immunization with SEA. ABR-214720 treatment was given as4 daily i.v. injections. For combination studies, docetaxel was injectedeither before completing TTS therapy or shortly after the lastABR-214720 injection.

Animals

Female C57B1/6 mice were used The mice were routinely used at the age of8 to 12 weeks and received food and water ad libitum.

Determination of Mouse Anti-SEA Antibodies

The concentration of anti-SEA antibodies in the mouse plasma samples wasdetermined by ELISA technique. The assay was performed with reagentaddition, incubation and washing in a sequential manner. In the firststep the microtiter plate wells were coated with 1 μg/mL (200 ng/well)of recombinant SEA in 50 mM NaHCO₃, pH 9.6. The wells were blocked with1% (w/v) bovine serum albumin (BSA) in 10 mM PBS, pH 7.4, 0.05% (w/v)Tween 20 (PBST). Samples and standards of affinity purified mouseanti-SEA antibodies were added, diluted in 1% BSA in PBST. A goatanti-mouse IgG antibody, was used as secondary antibody. Next, abiotinylated rabbit anti-goat IgG antibody was added. Thereafter,streptavidin conjugated with horseradish peroxidase was allowed to bindto the biotin groups of the tertiary antibody. The final step wasaddition of an enzyme substrate. The enzyme reaction was stopped by 1NH₂SO₄ and then the absorbance was monitored at 450 nm, with 650 nm as areference wavelength in an ELISA spectrophotometer. A four-parameterfunction was adjusted to the obtained concentration/absorbance values ofthe standards and the unknown concentration of anti-SEA antibodies insamples determined from standard curve. The measuring range wasestimated to 0.40-50 ng/mL. The sample dilution was at least 1:100, i.e.the limit of quantification (LOQ) in plasma was 40 ng/mL.

Induction of Anti-SEA Antibodies and Treatment with ChemotherapeuticAgents in C57B1/6 Mice

For induction of a primary anti-SEA response, mice were given 4intravenous injections of unconjugated SEA (3 μg/mouse/injection, in 0.2ml PBS containing 1% mouse serum) on days 1, 5, 9 and 13. Starting onday 29 after priming, ABR-214720 treatment was given as daily i.v.injections (10 μg/mouse/injection, in 0.2 ml PBS containing 1% mouseserum) on 4 consecutive days. Docetaxel was injected (1 or 2mg/mouse/injection i.p. in 0.2 ml 10.9-21.7% polysorbate 80, 5.1-10.2%ethanol and 0-0.45% NaCl) once during ABR-214720 treatment or aftercompletion of ABR-214720 treatment. At indicated time points bloodsamples were drained by vena saphena (day 26) and by vena cavaaspiration (day 46) for preparation of plasma. Eleven to seventeen micewere included in each experimental group.

Results

As illustrated in table 17, combination therapy was superior as comparedto treatment with single treatment TTS, in maintaining a low anti-SEAantibody titer. Single treatment TTS resulted in significant anti-SEAantibody titers. Docetaxel was administered during or after the TTStreatment cycle. It is clearly shown that the combination treatment bothduring as well as after the TTS cycle resulted in much lower secondaryresponse anti-SEA antibody titers as compared to the TTS treatment alone(groups 2-7 as compared to group 1).

TABLE 17 Inhibition of anti-SEA antibodies by combination withchemotherapy. Docetaxel Anti-SEA day 26 Anti-SEA day 46 Ratio Group SEA(i.v.) C215Fab-SEA (i.v.) 1 or 2 mg (i.p.) (ng/ml) (ng/ml)Secondary/primary No Days 1, 5, 9, 13 Days 29, 30, 31, 32 Day Primaryresponse Secondary response response 1 SEA C215Fab-SEA Vehicle, d 301290 27700 21.5 2 SEA C215Fab-SEA Docetaxel, d 29 (1 mg) 572 7350 12.8 3SEA C215Fab-SEA Docetaxel, d 30 (1 mg) 1290 23700 18.4 4 SEA C215Fab-SEADocetaxel, d 31 (1 mg) 898 4280 4.8 5 SEA C215Fab-SEA Docetaxel, d 32 (1mg) 813 4570 5.6 6 SEA C215Fab-SEA Docetaxel, d 33 (1 mg) 1240 12300 9.97 SEA C215Fab-SEA Docetaxel, d 33 (2 mg) 657 7340 11.2

Giving docetaxel treatment before integrated TTS injections or after TTSinjections resulted in lower secondary response anti-SEA antibody titersas compared to the TTS treatment alone. Thus, the combination of TTSwith chemotherapeutic agents for example docetaxel resulted in loweredlevels of anti-Superantigen (anti-Sag) antibodies after TTS treatmentcycles. Additional TTS treatment cycles can therefore be given withoutinterference from neutralizing high titer anti-Sag antibodies.

Example 17 Superantigen Combination Treatment Regimen for Monkeys

The animals (Cynomolgus monkey, purpose bred, approximately 15 monthsold and a weight of 2.5-3.2 kg) were allocated to treatment. Theindividual dose volumes were based on individual body weight.

The animals were given 5T4FabFab-SEA_(D227A) as intravenous (bolus)injections and gemcitabine as intravenous infusions (30 min). The totalduration of the study was 35 days. 5T4Fab-SEA_(D227A) was administeredas two treatment cycles, each of 5 days (Days 1-5 and 29-33) andgemcitabine during three occasions (Days 8, 15 and 22).

TABLE 18 Treatment: A sequential combination of 5T4Fab-SEA_(D227A)(i.v.) and gemcitabine (i.v. infusion during 30 min) to Cynomolgusmonkeys. Group Treatment 1 2 3 4 5T4Fab-SEA_(D227A) 18  0  6 18(μg/kg/occasion) Gemcitabine (mg/kg/occasion)  0 65 65 65 Animal No. andsex 761 M 757 M 759 M 755 M 766 F 762 F 764 F 760 FDetermination of the Concentration Anti-SEA Antibodies after CombinationTreatment Regimen

Blood samples were collected from a suitable vein (different from thatused for dosing) into tubes containing lithium heparin and immediatelyplaced in an ice-water bath or Cryorack. The blood samples were used todetermine concentration of anti-SEA antibodies.

An ELISA method was used to determine the concentration of anti-SEAantibodies in the plasma samples. The microtiter plate wells were coatedwith SEA in coating buffer. The wells were then blocked with bovineserum albumin in phosphate buffered saline containing Tween 20. Samplesand standards in the range 1-250 ng/mL, were added. Rabbit anti-monkeyIgG antibodies were used as the secondary antibody and in a subsequentstep a biotinylated swine anti-rabbit IgG antibody was added.Thereafter, streptavidin conjugated horseradish peroxidase was allowedto bind to the biotin groups of the tertiary antibody. The final stepwas addition of an enzyme substrate for horseradish peroxidase. Theenzyme reaction was stopped by addition of 2% oxalic acid and then theabsorbance was monitored at 405 nm in an ELISA spectrophotometer. Theunknown concentration of anti-SEA antibodies in the samples wasdetermined from the standard curve of affinity purified monkey anti-SEAIgG. The limit of quantification of the method was 0.8 μg/mL (5 μmol/mL)at a sample dilution of 1:100.

The results of the anti-SEA antibody levels in plasma are presented inTable 19. On Day 1, the anti-SEA antibody levels in all animals werebelow or slightly higher than the LOQ (0.80 mg/L). On Day 29 theanti-SEA antibody levels were elevated in the animals that received5T4Fab-SEA_(D227A) alone (Group 1). In Groups 3 and 4 (the sequentialcombination of 5T4Fab-SEA_(D227A) and gemcitabine), no changes or only aminor elevation in the anti-SEA antibody level was seen on Day 29. Thus,this showed that administration of gemcitabine in combination with TTSreduced the concentration of anti-SEA antibody production in the animalsfrom Day 1 to Day 29.

TABLE 19 Anti-SEA antibody levels in plasma sampled from Cynomolgusmonkeys after administration of 5T4Fab-SEA_(D227A) (Days 1-5, Days29-33) alone or in sequential combination with gemcitabine (Days 8, 15,22). Administration Anti-SEA antibody levels Group 5T4Fab- Animal No.(mg/L) No. SEA_(D227A) Gemcitabine and sex Day 1 Day 14 Day 21 Day 29 118 μg/kg — 761 M <LOQ 30.3 20.7  18.3 766 F 0.96 58.8 73.1  41.0 2 — 65mg/kg 757 M <LOQ <LOQ <LOQ <LOQ 762 F <LOQ <LOQ 0.87 <LOQ 3  6 μg/kg 65mg/kg 759 M <LOQ <LOQ <LOQ <LOQ 764 F <LOQ 22.0 8.04  6.86 4 18 μg/kg 65mg/kg 755 M <LOQ <LOQ <LOQ <LOQ 760 F 2.05 14.2 7.80  5.94Determination of the Concentration of TTS after Combination TreatmentRegimen

Blood samples were collected from a suitable vein (different from thatused for dosing) into tubes containing lithium heparin and immediatelyplaced in an ice-water bath or Cryorack. The blood samples were used todetermine concentration of 5T4Fab-SEA_(D227A).

An ELISA method was used to determine the concentration of5T4Fab-SEA_(D227A) in plasma samples. The method was based on the factthat one part of 5T4Fab-SEA_(D227A) is the Fab fragment of a monoclonalmouse antibody and the other part is the staphylococcal superantigenSEA.

Microtiter plates were coated with a goat anti-mouse kappa antibody in50 mM NaHCO₃, pH 9.6. The plates were washed in phosphate bufferedsaline containing Tween 20 between every incubation step. After blockingof the wells with bovine serum albumin, the diluted samples, andstandards in the range of 0.024-6.25 ng/mL, were added. The next stepwas addition of a biotinylated rabbit anti-SEA antibody. In thesubsequent step, horseradish peroxidase and streptavidin conjugated to adextran backbone was added. The final step was the addition ofsubstrate, TMB Peroxidase EIA substrate kit. The enzyme reaction wasstopped by addition of 1 N H₂SO₄, and then the absorbance at 450 nm wasmonitored by an ELISA spectrophotometer. The samples were quantifiedfrom the standard curve of 5T4Fab-SEA_(D227A).

The results from the bioanalysis of 5T4FAB-SEA_(D227A) concentration inplasma are presented in Table 20.

TABLE 20 Concentrations of 5T4Fab-SEA_(D227A) in plasma sampled fromCynomolgus monkeys after administration of 5T4Fab-SEA_(D227A) (Days 1-5,Days 29-33) alone or in sequential combination with gemcitabine (Days 8,15, 22). 5T4Fab-SEA_(D227A) 18 μg/kg 0 μg/kg 6 μg/kg 18 μg/kgGemcitabine 0 mg/kg 65 mg/kg 65 mg/kg 65 mg/kg Group 1 2 3 4 Animal no.and sex 761 M 766 F 757 M 762 F 759 M 764 F 755 M 760 F Day Time (h)Concentration of 5T4Fab-SEA_(D227A) (μg/L) in plasma 1 0 <LOQ <LOQ <LOQ<LOQ <LOQ <LOQ <LOQ <LOQ 0.083 385 399 <LOQ <LOQ 159 158 392 255 1 284309 n.a. n.a. 110 123 293 244 3 239 276 n.a. n.a. 70.6 89.0 204 231 897.0 74.9 n.a. n.a. 25.7 46.6 71.5 200 5 0 21.2 10.2 <LOQ <LOQ 3.42 32.210.7 255 0.083 418 399 <LOQ <LOQ 156 165 436 590 1 314 266 n.a. n.a. 118142 293 501 3 269 154 n.a. n.a. 73.2 112 213 434 8 94.8 57.6 n.a. n.a.24.7 70.4 64.9 398 29 0 <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 0.0830.82 <LOQ <LOQ <LOQ 144 84.8 433 236 1 <LOQ <LOQ n.a. n.a. 107 70.8 275215 3 <LOQ <LOQ n.a. n.a. 72.0 50.8 224 179 8 <LOQ <LOQ n.a. n.a. 24.930.1 70.8 126 33 0 <LOQ <LOQ <LOQ <LOQ 1.98 4.10 14.3 57.4 0.083 <LOQ<LOQ <LOQ <LOQ 97.1 48.3 449 542 1 <LOQ <LOQ n.a. n.a. 60.7 22.1 312 4993 <LOQ <LOQ n.a. n.a. 24.7 10.8 252 464 8 <LOQ <LOQ n.a. n.a. 6.23 5.0977.7 352 n.a. not analyzed

On Days 1 and 5 similar levels of 5T4Fab-SEA_(D227A) were achieved inall animals that received 5T4Fab-SEA_(D227A) (Groups 1, 3 and 4). OnDays 29 and 33, 5T4Fab-SEA_(D227A) was below LOQ (limit ofquantification, which is 0.5 μg/L) in the animals that received5T4Fab-SEA_(D227A) alone (Group 1). However on Days 29 and 33, the5T4Fab-SEA_(D227A) levels in the animals belonging to Groups 3 and 4were similar to the 5T4Fab-SEA_(D227A) levels found on Days 1 and 5.

Thus, the combination of TTS with chemotherapeutic agents for examplegemcitabine resulted in lowered levels of anti-Superantigen (anti-Sag)antibodies after TTS treatment cycles. Additional TTS treatment cyclescan therefore be given without interference from neutralizing high titeranti-Sag antibodies.

REFERENCES

All patents and publications mentioned in the specifications areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

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Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the invention asdefined by the appended sentences and descriptions. Moreover, the scopeof the present application is not intended to be limited to theparticular embodiments of the process, machine, manufacture, compositionof matter, means, methods and steps described in the specification. Asone will readily appreciate from the disclosure, processes, machines,manufacture, compositions of matter, means, methods, or steps, presentlyexisting or later to be developed that perform substantially the samefunction or achieve substantially the same result as the correspondingembodiments described herein may be utilized. Accordingly, the appendedstatements are intended to include within their scope such processes,machines, manufacture, compositions of matter, means, methods, or steps.

1. A method of reducing an antibody response to a tumor-targetedsuperantigen in a human, while enhancing a T cell response to atumor-targeted superantigen in a human and increasing the anti-tumoreffect of a chemotherapeutic agent in a human, comprising the steps of:reducing an antibody response to a tumor-targeted superantigen whenadministered to a human, which antibody response is reduced comparedwith the antibody response to a tumor-targeted superantigen whenadministered to a human without a chemotherapeutic agent, whileenhancing a T cell response to a tumor-targeted superantigen whenadministered to a human, which T cell response is enhanced compared withthe T cell response to a tumor-targeted superantigen when administeredwithout a chemotherapeutic agent, and increasing the anti-tumor effectof a chemotherapeutic agent when administered to a human, whichanti-tumor effect is increased compared with the anti-tumor effect of achemotherapeutic agent when administered to a human without atumor-targeted superantigen, by administering non-localized via thecirculatory system to the human a tumor-targeted superantigen comprisingthe modified superantigen SEA/E-120 conjugated with an antibodytargeting moiety specific for a tumor-associated cell surface antigen,and not also administering intratumorally or intrathecally to said humansaid tumor-targeted superantigen, and administering a chemotherapeuticagent.
 2. The method of claim 1, wherein the antibody targeting moietyspecific for a tumor-associated cell surface antigen is selected fromthe group consisting of full length antibodies, Fv, Fab, F(ab)₂, andsingle chain antibodies.
 3. The method of claim 2, wherein the modifiedsuperantigen comprises the amino acid sequence of SEQ. ID. NO. 3 andwherein the modified superantigen is covalently conjugated to theantibody targeting moiety.
 4. The method of claim 3, wherein theantibody targeting moiety is specific for the 5T4 or EpCAM/GA733-2tumor-associated cell surface antigens.
 5. The method of claim 3,wherein the antibody targeting moiety specific for a tumor-associatedcell surface antigen is 5T4Fab or C215Fab.
 6. The method of claim 3,wherein the chemotherapeutic agent is a cytostatic drug.
 7. The methodof claim 6, wherein the cytostatic drug is an alkylating agent selectedfrom the group consisting of busulfan, chlorambucil, cyclophosphamide,melphalan, carmustine, and lomustine; an antimetabolite selected fromthe group consisting of 5-fluorouracil, gemcitabine, and pemetrexed; anantitumor antibiotic selected from the group consisting of doxorubicin,daunorubicin, mitomycin, actinomycin D, and bleomycin; a mitoticinhibitor selected from the group consisting of paclitaxel, docetaxel,vinblastine, vincristine, and etoposide; or a platimun based compoundselected from the group consisting of cisplatin, carboplatin, andoxaliplatin.
 8. The method of claim 3, wherein the human has a cancerselected from the group consisting of lung, breast, colon, kidney,pancreatic, ovarian, stomach, cervix and prostate cancer.
 9. The methodof claim 3, wherein the tumor-targeted superantigen is administeredprior to the administration of the chemotherapeutic agent, after theadministration of the chemotherapeutic agent, during the administrationof the chemotherapeutic agent, or between administration of thechemotherapeutic agent.
 10. A method of reducing an antibody response toa tumor-targeted superantigen in a human, and enhancing a T cellresponse to a tumor-targeted superantigen in a human, comprising thesteps of: reducing an antibody response to a tumor-targeted superantigenwhen administered to a human, which antibody response is reducedcompared with the antibody response to a tumor-targeted superantigenwhen administered to a human without a chemotherapeutic agent, andenhancing a T cell response to a tumor-targeted superantigen whenadministered to a human, which T cell response is enhanced compared withthe T cell response to a tumor-targeted superantigen when administeredwithout a chemotherapeutic agent by administering non-localized via thecirculatory system to the human a tumor-targeted superantigen comprisingthe modified superantigen SEA/E-120 conjugated with an antibodytargeting moiety specific for a tumor-associated cell surface antigen,and not also administering intratumorally or intrathecally to said humansaid tumor-targeted superantigen, and administering a chemotherapeuticagent.
 11. The method of claim 10, wherein the antibody targeting moietyspecific for a tumor-associated cell surface antigen is selected fromthe group consisting of full length antibodies, Fv, Fab, F(ab)₂, andsingle chain antibodies.
 12. The method of claim 11, wherein themodified superantigen comprises the amino acid sequence of SEQ. ID. NO.3 and wherein the modified superantigen is covalently conjugated to theantibody targeting moiety.
 13. The method of claim 12, wherein theantibody targeting moiety is specific for the 5T4 or EpCAM/GA733-2tumor-associated cell surface antigens.
 14. The method of claim 12,wherein the antibody targeting moiety specific for a tumor-associatedcell surface antigen is 5T4Fab or C215Fab.
 15. The method of claim 12,wherein the chemotherapeutic agent is a cytostatic drug.
 16. The methodof claim 15, wherein the cytostatic drug is an alkylating agent selectedfrom the group consisting, of busulfan, chlorambucil, cyclophosphamide,melphalan, carmustine, and lomustine; an antimetabolite selected fromthe group consisting of 5-fluorouracil, gemcitabine, and pemetrexed; anantitumor antibiotic selected from the group consisting of doxorubicin,daunorubicin, mitomycin, actinomycin D, and bleomycin; a mitoticinhibitor selected from the group consisting of paclitaxel, docetaxel,vinblastine, vincristine, and etoposide; or a platimun based compoundselected from the group consisting of cisplatin, carboplatin, andoxaliplatin.
 17. The method of claim 12, wherein the human has a cancerselected from the group consisting of lung, breast, colon, kidney,pancreatic, ovarian, stomach, cervix and prostate cancer.
 18. The methodof claim 12, wherein the tumor-targeted superantigen is administeredprior to the administration of the chemotherapeutic agent, after theadministration of the chemotherapeutic agent, during the administrationof the chemotherapeutic agent, or between administration of thechemotherapeutic agent.
 19. A method of reducing an antibody response toa tumor-targeted superantigen in a human, and increasing the anti-tumoreffect of a chemotherapeutic agent in a human, comprising the steps ofreducing an antibody response to a tumor-targeted superantigen whenadministered to a human, which antibody response is reduced comparedwith the antibody response to a tumor-targeted superantigen whenadministered to a human without a chemotherapeutic agent, and increasingthe anti-tumor effect of a chemotherapeutic agent when administered to ahuman, which anti-tumor effect is increased compared with the anti-tumoreffect of a chemotherapeutic agent when administered to a human withouta tumor-targeted superantigen by administering non-localized via thecirculatory system to the human a tumor-targeted superantigen comprisingthe modified superantigen SEA/E-120 conjugated with an antibodytargeting moiety specific for a tumor-associated cell surface antigen,and not also administering intratumorally or intrathecally to said humansaid tumor-targeted superantigen, and administering a chemotherapeuticagent.
 20. The method of claim 19, wherein the antibody targeting moietyspecific for a tumor-associated cell surface antigen is selected fromthe group consisting of full length antibodies, Fv, Fab, F(ab)₂, andsingle chain antibodies.
 21. The method of claim 20, wherein themodified superantigen comprises the amino acid sequence of SEQ. ID. NO.3 and wherein the modified superantigen is covalently conjugated to theantibody targeting moiety.
 22. The method of claim 21, wherein theantibody targeting moiety is specific for the 5T4 or EpCAM/GA733-2tumor-associated cell surface antigens.
 23. The method of claim 21,wherein the antibody targeting moiety specific for a tumor-associatedcell surface antigen is 5T4Fab or C215Fab.
 24. The method of claim 21,wherein the chemotherapeutic agent is a cytostatic drug.
 25. The methodof claim 24, wherein the cytostatic drug is an alkylating agent selectedfrom the group consisting of busulfan, chlorambucil, cyclophosphamide,melphalan, carmustine, and lomustine; an antimetabolite selected fromthe group consisting of 5-fluorouracil, gemcitabine, and pemetrexed; anantitumor antibiotic selected from the group consisting of doxorubicin,daunorubicin, mitomycin, actinomycin D, and bleomycin; a mitoticinhibitor selected from the group consisting of paclitaxel, docetaxel,vinblastine, vincristine, and etoposide; or a platimun based compoundselected from the group consisting of cisplatin, carboplatin, andoxaliplatin.
 26. The method of claim 21, wherein the human has a cancerselected from the group consisting of lung, breast, colon, kidney,pancreatic, ovarian, stomach, cervix and prostate cancer.
 27. The methodof claim 21, wherein the tumor-targeted superantigen is administeredprior to the administration of the chemotherapeutic agent, after theadministration of the chemotherapeutic agent, during the administrationof the chemotherapeutic agent, or between administration of thechemotherapeutic agent.
 28. The method of claim 24, wherein thecytotoxic drug is docetaxel.
 29. The method of claim 24, wherein thecytotoxic drug is gemcitabine.